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Tomoyuki Numata Last modified date：2023.11.27

Associate Professor / Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences / Department of Bioscience and Biotechnology / Faculty of Agriculture


Papers
1. Numata Tomoyuki, Kashiba Tohru, Hino Madoka, FUNATSU Gunki, ISHIGURO Masatune, YAMASAKI Nobuyuki, KIMURA Makoto, Expression and Mutational Analysis of Amino Acid Residues Involved in Catalytic Activity in a Ribonuclease MC1 from the Seeds of Bitter Gourd, Bioscience, biotechnology, and biochemistry, 10.1271/bbb.64.603, 64, 3, 603-605, 2000.03, The ribonuclease MC1 (RNase MC1) from seeds of bitter gourd (Momordica charantia) consists of 190 amino acids and belongs to the RNase T2 family, including fungal RNase typified by RNase Rh from Rhizopus niveus. We expressed RNase MC1 in Escherichia coli cells and made use of site-directed mutagenesis to identify essential amino acid residues for catalytic activity. Mutations of His34 and His88 to Ala completely abolished the enzymatic activity, and considerable decreases in the enzymatic activity were observed in cases of mutations of His83,Glu84,and Lys87,when yeast RNA was used as a substrate. Kinetic parameters for the enzymatic activity of the mutants of His83,Glu84,and Lys87 were analyzed using a dinucleoside monophosphate CpU. K_m values for the mutants were approximately like that for wild-type, while k_ values were decreased by about 6 to 25-fold. These results suggest that His34,His83,Glu84,Lys87,and His88 in RNase MC1 may be involved in the catalytic function. These observation suggests that RNase MC1 from a plant catalyzes RNA degradation in a similar manner to that of fungal RNases..
2. Akio Suzuki, Min Yao, Isao Tanaka, Tomoyuki Numata, Singo Kikukawa, Nobuyuki Yamasaki, Makoto Kimura, Crystal structures of the ribonuclease MC1 from bitter gourd seeds, complexed with 2'-UMP or 3'-UMP, reveal structural basis for uridine specificity, Biochem.Biophys.Res.Commun., 10.1006/bbrc.2000.3318, 275, 572-576, 2000.08.
3. Tomoyuki Numata, Akio Suzuki, Min Yao, Isao Tanaka, Makoto Kimura, Amino acid residues in ribonuclease MC1 from bitter gourd seeds which are essential for uridine specificity, Biochemistry, 10.1021/bi002096f, 40, 524-530, 2001.01.
4. T Numata, M Kimura, Contribution of Gln9 and Phe80 to substrate binding in ribonuclease MC1 from bitter gourd seeds, JOURNAL OF BIOCHEMISTRY, 130, 5, 621-626, 2001.11, Ribonuclease MC1 (RNase MC1) isolated from bitter gourd (Momordica charantia) seeds specifically cleaves phosphodiester bonds on the 5'-side of uridine. The crystal structures of RNase MC1 in complex with 2'-UMP or 3'-UMP reveal that Gln9, Asn71, Leu73, and Phe80 are involved in uridine binding by hydrogen bonding and hydrophobic interactions [Suzuki et al. (2000) Biochem. Biophys. Res. Commun. 275, 572-576]. To evaluate the contribution of Gln9 and Phe80 to uridine binding, Gln9 was replaced with Ala, Phe, Glu, or His, and Phe80 with Ala by site-directed mutagenesis. The kinetic properties of the resulting mutant enzymes were characterized using cytidylyl-3',5'-uridine (CpU) as a substrate. The mutant Q9A exhibited a 3.7-fold increased K-m and 27.6-fold decreased k(cat), while three other mutations, Q9F, Q9E, and Q9H, predominantly affected the k(cat) value. Replacing Phe80 with Ala drastically reduced the catalytic efficiency (k(cat)/K-m) with a minimum K-m value equal to 8 mM. It was further found that the hydrolytic activities of the mutants toward cytidine-2',3'-cyclic monophosphate (cCMP) were reduced. These results demonstrate that Gln9 and Phe80 play essential roles not only in uridine binding but also in hydrolytic activity. Moreover, we produced double Ala substituted mutants at Gln9, Asn71, Leu73, and Phe80, and compared their kinetic properties with those of the corresponding single mutants. The results suggest that these four residues may contribute to uridine binding in a mutually independent manner..
5. Y Kouzuma, M Mizoguchi, H Takagi, H Fukuhara, M Tsukamoto, T Numata, M Kimura, Reconstitution of archaeal ribonuclease P from RNA and four protein components, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/S0006-291X(03)01034-9, 306, 3, 666-673, 2003.07, Ribonuclease P (RNase P) is an endonuclease responsible for generating the 5' end of matured tRNA molecules. A homology search of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 genome database revealed that the four genes, PH1481, PH1601, PH1771, and PH1877, have a significant homology to those encoding RNase P protein subunits, hpop5, Rpp21, Rpp29, and Rpp30, of human, respectively. These genes were expressed in Escherichia coli cells, and the resulting proteins Ph1481p, Ph1601p, Ph1771p, and Ph1877p were purified to apparent homogeneity in a set of column chromatographies. The four proteins were characterized in terms of their capability to bind the cognate RNase P RNA from P. horikoshii. All four proteins exhibited the binding activity to the RNase P RNA. In vitro reconstitution of four putative RNase P proteins with the in vitro transcripted P. horikoshii RNase P RNA revealed that three proteins Ph1481p, Ph1601p, and Ph1771p, and RNase P RNA are minimal components for the RNase P activity. However, addition of the fourth protein Ph1877p strongly stimulated enzymatic activity, indicating that all four proteins and RNase P RNA are essential for optimal RNase P activity. The present data will pave the way for the elucidation of the reaction mechanism for archaeal as well as eukaryotic RNase P. (C) 2003 Elsevier Science (USA). All rights reserved..
6. T Numata, A Suzuki, Y Kakuta, K Kimura, M Yao, Tanaka, I, Y Yoshida, T Ueda, M Kimura, Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5 '-GMP: Structural basis for alterations in substrate specificity, BIOCHEMISTRY, 10.1021/bi034103g, 42, 18, 5270-5278, 2003.05, Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5'GMP, were determined at resolutions higher than 2 A. In the N71T-5'-GMP and N71S-5'-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC 1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 finely by changing the size and shape of the B2 site..
7. H Takagi, M Watanabe, Y Kakuta, R Kamachi, T Numata, Tanaka, I, M Kimura, Crystal structure of the ribonuclease P protein Ph1877p from hyperthermophilic archaeon Pyrococcus horikoshii OT3, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2004.05.055, 319, 3, 787-794, 2004.07, Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of pre-tRNA. Protein Ph1877p is one of essential components of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 RNase P [Biochem. Biophys. Res. Commun. 306 (2003) 666]. The crystal structure of Ph1877p was determined at 1.8 Angstrom by X-ray crystallography and refined to a crystallographic R factor of 22.96% (R-free of 26.77%). Ph1877p forms a TIM barrel structure, consisting of ten alpha-helices and seven beta-strands, and has the closest similarity to the TIM barrel domain of Escherichia coli cytosine deaminase with a root-mean square deviation of 3.0 Angstrom. The protein Ph1877p forms an oblate ellipsoid, approximate dimensions being 45 Angstrom x 43 Angstrom x 39 Angstrom, and the electrostatic representation indicated the presence of several clusters of positively charged amino acids present on the molecular surface. We made use of site-directed mutagenesis to assess the role of twelve charged amino acids, Lys42, Arg68, Arg87, Arg90, Asp98, Arg107, His114, Lys123, Lys158, Arg176, Asp180, and Lys196 related to the RNase P activity. Individual mutations of Arg90, Arg107, Lys123, Arg176, and Lys196 by Ala resulted in reconstituted particles with reduced enzymatic activities (32-48%) as compared with that reconstituted RNase P by wild-type Ph1877p. The results presented here provide an initial step for definite understanding of how archaeal and eukaryotic RNase Ps mediate substrate recognition and process 5'-leader sequence of pre-tRNA. (C) 2004 Elsevier Inc. All rights reserved..
8. T Numata, Ishimatsu, I, Y Kakuta, Tanaka, I, M Kimura, Crystal structure of archaeal ribonuclease P protein Ph1771 p from Pyrococcus horikoshii OT3: An archaeal homolog of eukaryotic ribonuclease P protein Rpp29, RNA-A PUBLICATION OF THE RNA SOCIETY, 10.1261/rna.7560904, 10, 9, 1423-1432, 2004.09, Ribonuclease P (RNase P) is the endonuclease responsible for the removal of 5' leader sequences from tRNA precursors. The crystal structure of an archaeal RNase P protein, Ph1771p (residues 36-127) from hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined at 2.0 Angstrom resolution by X-ray crystallography. The structure is composed of four helices (alpha1-alpha4) and a six-stranded antiparallel beta-sheet (beta1-beta6) with a protruding beta-strand (beta7) at the C-terminal region. The strand beta7 forms an antiparallel beta-sheet by interacting with strand beta4 in a symmetry-related molecule, suggesting that strands beta4 and beta7 could be involved in protein-protein interactions with other RNase P proteins. Structural comparison showed that the beta-barrel structure of Ph1771p has a topological resemblance to those of Staphylococcus aureus translational regulator Hfq and Haloarcula marismortui ribosomal protein L21E, suggesting that these RNA binding proteins have a common ancestor and then diverged to specifically bind to their cognate RNAs. The structure analysis as well as structural comparison suggested two possible RNA binding sites in Ph1771p, one being a concave surface formed by terminal alpha-helices (alpha1-alpha4) and beta-strand beta6, where positively charged residues are clustered. A second possible RNA binding site is at a loop region connecting strands beta2 and beta3, where conserved hydrophilic residues are exposed to the solvent and interact specifically with sulfate ion. These two potential sites for RNA binding are located in close proximity. The crystal structure of Ph1771p provides insight into the structure and function relationships of archaeal and eukaryotic RNase P..
9. K Kimura, T Numata, Y Kakuta, M Kimura, Amino acids conserved at the C-terminal half of the ribonuclease T2 family contribute to protein stability of the enzymes, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 10.1271/bbb.68.1748, 68, 8, 1748-1757, 2004.08, The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr(101), Phe(102), Ala(105), and Phe(190) resulted in a significant decrease in themostability; the T-m values were 47-58degreesC compared to that for the wild type (64degreesC). Mutations of Pro(125), Gly(127), Gly(144), and Val(165) caused a moderate decrease in thermostability (T-m: 60-62degreesC). In contrast, mutations of Asp(107) and Gly(173) did little effect on thermostability. The contribution of Tyr(101), Phe(102), Pro(125), and Gly(127) to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr101, Phe102, Ala 105, Pro125, Gly127, Gly(144), Leu(162), Val(161), and Phe(190) conserved in the RNase T2 family play an important role in the stability of the proteins..
10. Y Kakuta, Ishimatsu, I, T Numata, K Kimura, M Yao, Tanaka, I, M Kimura, Crystal structure of a ribonuclease P protein Ph1601p from Pyrococcus horikoshii OT3: An archaeal homologue of human nuclear ribonuclease P protein Rpp21, BIOCHEMISTRY, 10.1021/bi050738z, 44, 36, 12086-12093, 2005.09, Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the removal of 5' leader sequences from tRNA precursors (pre-tRNA). The human protein Rpp21 is essential for human RNase P activity in tRNA processing in vitro. The crystal structure of Ph1601p from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, the archaeal homologue of Rpp21, was determined using the multiple anomalous dispersion (MAD) method with the aid of anomalous scattering in zinc and selenium at 1.6 angstrom resolution. Ph1601p comprises an N-terminal domain (residues 1-55), a central linker domain (residues 56-79), and a C-terminal domain (residues 80-120), forming an L-shaped structure. The N-terminal domain consists of two long alpha-helices, while the central and C-terminal domains fold in a zinc ribbon domain. The electrostatic potential representation indicates the presence of positively charged clusters along the L arms, suggesting a possible role in RNA binding. A single zinc ion binds the well-ordered binding site that consists of four Cys residues (Cys68, Cys71, Cys97, and Cys100) and appears to stabilize the relative positions of the N- and C-domains. Mutations of Cys68 and Cys71 or Cys97 and Cys100 to Ser destabilize the protein structure, which results in inactivation of the RNase P activity. In addition, site-directed mutagenesis suggests that Lys69 at the central loop and Arg86 and Arg105 at the zinc ribbon domain are strongly involved in the functional activity, while Arg22, Tyr-44, Arg65, and Arg84 play a modest role in the activity..
11. H Oshikane, K Sheppard, S Fukai, Y Nakamura, R Ishitani, T Numata, RL Sherrer, L Feng, E Schmitt, M Panvert, S Blanquet, Y Mechulam, D Soll, O Nureki, Structural basis of RNA-dependent recruitment of glutamine to the genetic code, SCIENCE, 10.1126/science.1128470, 312, 5782, 1950-1954, 2006.06, Glutaminyl-transfer RNA (Gln-tRNA(Gln)) in archaea is synthesized in a pretranslational amidation of misacylated Glu-tRNA(Gln) by the heterodimeric Glu-tRNA(Gln) amidotransferase GatDE. Here we report the crystal structure of the Methanothermobacter thermautotrophicus GatDE complexed to tRNA(Gln) at 3.15 angstroms resolution. Biochemical analysis of GatDE and of tRNAGln mutants characterized the catalytic centers for the enzyme's three reactions (glutaminase, kinase, and amidotransferase activity). A 40 angstrom-long channel for ammonia transport connects the active sites in GatD and GatE. tRNA(Gln) recognition by indirect readout based on shape complementarity of the D loop suggests an early anticodon-independent RNA-based mechanism for adding glutamine to the genetic code..
12. T Numata, S Fukai, Y Ikeuchi, T Suzuki, O Nureki, Structural basis for sulfur relay to RNA mediated by heterohexameric TusBCD complex, STRUCTURE, 10.1016/j.str.2005.11.009, 14, 2, 357-366, 2006.02, Uridine at wobble position 34 of tRNA(Lys), tRNA(Glu), and tRNA Gin is exclusively modified into 2-thiouridine (s(2)U), which is crucial for both precise codon recognition and recognition by the cognate aminoacyl-tRNA synthetases. Recent Escherichia coli genetic studies revealed that the products of five novel genes, tusABCDE, function in the (SU)-U-2 modification. Here, we solved the 2.15 angstrom crystal structure of the E. coli TusBCD complex, a sulfur transfer mediator, forming a heterohexamer composed of a dimer of the hetero-trimer. Structure-based sequence alignment suggested two putative active site Cys residues, Cys79 (in TusC) and Cys78 (in TusD), which are exposed on the hexameric complex. In vivo mutant analyses revealed that only Cys78, in the TusD subunit, participates in sulfur transfer during the s(2)U modification process. Since the single Cys acts as a catalytic residue, we proposed that TusBCD mediates sulfur relay via a putative persulfide state of the TusD subunit..
13. Tomoyuki Numata, Yoshiho Ikeuchi, Shuya Fukai, Tsutomu Suzuki, Osamu Nureki, Snapshots of tRNA sulphuration via an adenylated intermediate, NATURE, 10.1038/nature04896, 442, 7101, 419-424, 2006.07.
14. T Tsukazaki, H Mori, S Fukai, T Numata, A Perederina, H Adachi, H Matsumura, K Takano, S Murakami, T Inoue, Y Mori, T Sasaki, DG Vassylyev, O Nureki, K Ito, Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 10.1107/S1744309106007779, 62, Pt 4, 376-380, 2006.04, Thermus thermophilus has a multi-path membrane protein, TSecDF, as a single-chain homologue of Escherichia coli SecD and SecF, which form a translocon-associated complex required for efficient preprotein translocation and membrane-protein integration. Here, the cloning, expression in E. coli, purification and crystallization of TSecDF are reported. Overproduced TSecDF was solubilized with dodecylmaltoside, chromatographically purified and crystallized by vapour diffusion in the presence of polyethylene glycol. The crystals yielded a maximum resolution of 4.2 angstrom upon X-ray irradiation, revealing that they belonged to space group P4(3)2(1)2. Attempts were made to improve the diffraction quality of the crystals by combinations of micro-stirring, laser-light irradiation and dehydration, which led to the eventual collection of complete data sets at 3.74 angstrom resolution and preliminary success in the single-wavelength anomalous dispersion analysis. These results provide information that is essential for the determination of the three-dimensional structure of this important membrane component of the protein-translocation machinery..
15. T Numata, Y Ikeuchi, S Fukai, H Adachi, H Matsumura, K Takano, S Murakami, T Inoue, Y Mori, T Sasaki, T Suzuki, O Nureki, Crystallization and preliminary X-ray analysis of the tRNA thiolation enzyme MnmA from Escherichia coli complexed with tRNA(Glu), ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 10.1107/S174430910600738X, 62, Pt 4, 368-371, 2006.04, MnmA catalyzes a sulfuration reaction to synthesize 2-thiouridine at the wobble positions of tRNA(Glu), tRNA(Gln) and tRNA(Lys) in Escherichia coli. The binary complex of MnmA and tRNA(Glu) was crystallized in two different crystal forms: forms I and II. Cocrystallization of MnmA - tRNA(Glu) with ATP yielded form III crystals. The three crystal forms diffracted to 3.1, 3.4 and 3.4 angstrom resolution, respectively, using synchrotron radiation at SPring-8. These crystals belong to space groups C2, I2(1)2(1)2(1) and C2, with unit-cell parameters a = 225.4, b = 175.8, c = 53.0 angstrom, beta = 101.6 degrees, a = 101.5, b = 108.0, c = 211.2 angstrom and a = 238.1, b = 102.1, c = 108.2 angstrom, beta = 117.0 degrees, respectively. The asymmetric units of these crystals are expected to contain two, one and two MnmA - tRNA(Glu) complexes, respectively..
16. H Fukuhara, M Kifusa, M Watanabe, A Terada, T Honda, T Numata, Y Kakuta, M Kimura, A fifth protein subunit Ph1496p elevates the optimum temperature for the ribonuclease P activity from Pyrococcus horikoshii OT3, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2006.02.192, 343, 3, 956-964, 2006.05, Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5' leader sequence of precursor tRNA. We previously found that the reconstituted particle (RP) composed of RNase P RNA and four proteins (Ph1481p, Ph1601p, Ph1771p, and Ph1877p) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 exhibited the RNase P activity, but had a lower optimal temperature (around at 55 degrees C), as compared with 70 degrees C of the authentic RNase P from P. horikoshii [Kouzuma et A., Biochem. Biophys. Res. Commun. 306 (2003) 666-673]. In the present study, we found that addition of a fifth protein Ph1496p, a putative ribosomal protein L7Ae, to RP specifically elevated the optimum temperature to about 70 degrees C comparable to that of the authentic RNase P. Characterization using get shift assay and chemical probing localized Ph1496p binding sites on two stem-loop structures encompassing nuclecitides A116-G201 and G229-C276 in P. horikoshii RNase P RNA. Moreover, the crystal structure of Ph1496p was determined at 2.0 angstrom resolution by the molecular replacement method using ribosomal protein L7Ae from Haloarcula marismortui as a search model. Ph1496p comprises five of.-helices and a four stranded beta-shect. The beta-sheet is sandwiched by three helices (alpha 1, alpha 4, and alpha 5) at one side and two helices (alpha 2 and alpha 3) at other side. The archaeal ribosomal protein L7Ae is known to be a triple functional protein, serving as a protein component in ribosome and ribonucleoprotein complexes, box C/D, and box H/ACA. Although we have at present no direct evidence that Ph1496p is a real protein component in the P. horikoshii RNase P, the present result may assign an RNase P protein to L7Ae as a fourth function. (c) 2006 Elsevier Inc. All rights reserved..
17. Yukimatsu Toh, Tomoyuki Numata, Kazunori Watanabe, Daijiro Takeshita, Osamu Nureki, Kozo Tomita, Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme, EMBO JOURNAL, 10.1038/emboj.2008.124, 27, 14, 1944-1952, 2008.07, CCA-adding enzyme builds the 3'-end CCA of tRNA without a nucleic acid template. The mechanism for the maintenance of fidelity during the CCA-adding reaction remains elusive. Here, we present almost a dozen complex structures of the class I CCA-adding enzyme and tRNA mini-helices (mini-D(73)N(74), mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75); D(73) is a discriminator nucleotide and N is either A, G, or U). The mini-D(73)N(74) complexes adopt catalytically inactive open forms, and CTP shifts the enzymes to the active closed forms and allows N(74) to flip for CMP incorporation. In contrast, unlike the catalytically active closed form of the mini-D(73)C(74)C(75) complex, the mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75) complexes adopt inactive open forms. Only the mini-D(73)C(74)U(75) accepts AMP to a similar extent as mini-D(73)C(74)C(75), and ATP shifts the enzyme to a closed, active form and allows U(75) to flip for AMP incorporation. These findings suggest that the 3'-region of RNA is proofread, after two nucleotide additions, in the closed, active form of the complex at the AMP incorporation stage. This proofreading is a prerequisite for the maintenance of fidelity for complete CCA synthesis..
18. Yukimatsu Toh, Daijiro Takeshita, Tomoyuki Numata, Shuya Fukai, Osamu Nureki, Kozo Tomita, Mechanism for the definition of elongation and termination by the class II CCA-adding enzyme, EMBO JOURNAL, 10.1038/emboj.2009.260, 28, 21, 3353-3365, 2009.11, The CCA-adding enzyme synthesizes the CCA sequence at the 3' end of tRNA without a nucleic acid template. The crystal structures of class II Thermotoga maritima CCA-adding enzyme and its complexes with CTP or ATP were determined. The structure-based replacement of both the catalytic heads and nucleobase-interacting neck domains of the phylogenetically closely related Aquifex aeolicus A-adding enzyme by the corresponding domains of the T. maritima CCA-adding enzyme allowed the A-adding enzyme to add CCA in vivo and in vitro. However, the replacement of only the catalytic head domain did not allow the A-adding enzyme to add CCA, and the enzyme exhibited (A, C)-adding activity. We identified the region in the neck domain that prevents (A, C)-adding activity and defines the number of nucleotide incorporations and the specificity for correct CCA addition. We also identified the region in the head domain that defines the terminal A addition after CC addition. The results collectively suggest that, in the class II CCA-adding enzyme, the head and neck domains collaboratively and dynamically define the number of nucleotide additions and the specificity of nucleotide selection. The EMBO Journal (2009) 28, 3353-3365. doi: 10.1038/emboj.2009.260; Published online 10 September 2009.
19. Takuo Osawa, Hideko Inanaga, Tomoyuki Numata, Crystallization and preliminary X-ray diffraction analysis of the tRNA-modification enzyme GidA from Aquifex aeolicus, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 10.1107/S1744309109013591, 65, Pt 5, 508-511, 2009.05, The 5-carboxymethylaminomethyl modification of uridine at the first position of the tRNA anticodon is crucial for accurate protein synthesis by stabilizing the correct codon-anticodon pairing on the ribosome. Two conserved enzymes, GidA and MnmE, are involved in this modification process. Aquifex aeolicus GidA was crystallized in two different crystal forms: forms I and II. These crystals diffracted to 3.2 and 2.3 angstrom resolution, respectively, using synchrotron radiation at the Photon Factory. These crystals belonged to space groups I2(1)2(1)2(1) and P2(1) with unit-cell parameters a = 101.6, b = 213.3, c = 231.7 angstrom and a = 119.4, b = 98.0, c = 129.6 angstrom, beta = 90.002 degrees, respectively. The asymmetric units of these crystals are expected to contain two and four molecules, respectively..
20. Takuo Osawa, Koichi Ito, Hideko Inanaga, Osamu Nureki, Kozo Tomita, Tomoyuki Numata, Conserved Cysteine Residues of GidA Are Essential for Biogenesis of 5-Carboxymethylaminomethyluridine at tRNA Anticodon, STRUCTURE, 10.1016/j.str.2009.03.013, 17, 5, 713-724, 2009.05, The 5-carboxymethylaminomethyl modification of uridine (cmnm(5)U) at the anticodon first position occurs in tRNAs that read split codon boxes ending with purine. This modification is crucial for correct translation, by restricting codon-anticodon wobbling. Two conserved enzymes, GidA and MnmE, participate in the cmnm(5)U modification process. Here we determined the crystal structure of Aquifex aeolicus GidA at 2.3 angstrom resolution. The structure revealed the tight interaction of GidA with FAD. Structure-based mutation analyses allowed us to identify two conserved Cys residues in the vicinity of the FAD-binding site that are essential for the cmnm(5)U modification in vivo. Together with mutational analysis of MnmE, we propose a mechanism for the cmnm(5)U modification process where GidA, but not MnmE, attacks the C6 atom of uridine by a mechanism analogous to that of thymidylate synthase. We also present a tRNA-docking model that provides structural insights into the tRNA recognition mechanism for efficient modification..
21. Takayuki Ohnuma, Takuo Osawa, Tamo Fukamizo, Tomoyuki Numata, Crystallization and preliminary X-ray diffraction analysis of a class V chitinase from Nicotiana tabacum, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 10.1107/S1744309110039060, 66, Pt 12, 1599-1601, 2010.12, The plant chitinases, which have been implicated in self-defence against pathogens, are divided into at least five classes (classes I, II, III, IV and V). Although the crystal structures of several plant chitinases have been solved, no crystal structure of a class V chitinase has been reported to date. Here, the crystallization of Nicotiana tabacum class V chitinase (NtChiV) using the vapour-diffusion method is reported. The NtChiV crystals diffracted to 1.2 A resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 62.4, b = 120.3, c = 51.9 A. The asymmetric unit of the crystals is expected to contain one molecule..
22. Yoshiho Ikeuchi, Satoshi Kimura, Tomoyuki Numata, Daigo Nakamura, Takashi Yokogawa, Toshihiko Ogata, Takeshi Wada, Takeo Suzuki, Tsutomu Suzuki, Agmatine-conjugated cytidine in a tRNA anticodon is essential for AUA decoding in archaea, NATURE CHEMICAL BIOLOGY, 10.1038/NCHEMBIO.323, 6, 4, 277-282, 2010.04, A modified base at the first (wobble) position of some tRNA anticodons is critical for deciphering the genetic code. In eukaryotes and eubacteria, AUA codons are decoded by tRNAs(Ile) with modified bases pseudouridine (and/or inosine) and lysidine, respectively. The mechanism by which archaeal species translate AUA codons is unclear. We describe a polyamine-conjugated modified base, 2-agmatinylcytidine (agm(2)C or agmatidine), at the wobble position of archaeal tRNA(Ile) that decodes AUA codons specifically. We demonstrate that archaeal cells use agmatine to synthesize agm(2)C of tRNA(Ile). We also identified a new enzyme, tRNA(Ile)-agm(2)C synthetase (TiaS), that catalyzes agm(2)C formation in the presence of agmatine and ATP. Although agm2C is chemically similar to lysidine, TiaS constitutes a distinct class of enzyme from tRNA(Ile)-lysidine synthetase (TilS), suggesting that the decoding systems evolved convergently across domains..
23. Takuo Osawa, Satoshi Kimura, Naohiro Terasaka, Hideko Inanaga, Tsutomu Suzuki, Tomoyuki Numata, Structural basis of tRNA agmatinylation essential for AUA codon decoding, NATURE STRUCTURAL & MOLECULAR BIOLOGY, 10.1038/nsmb.2144, 18, 11, 1275-U123, 2011.11, The cytidine at the first position of the anticodon (C34) in the AUA codon-specific archaeal tRNA(Ile2) is modified to 2-agmatinylcytidine (agm(2)C or agmatidine), an agmatine-conjugated cytidine derivative, which is crucial for the precise decoding of the genetic code. Agm(2)C is synthesized by tRNA(Ile)-agm(2)C synthetase (TiaS) in an ATP-dependent manner. Here we present the crystal structures of the Archaeoglobus fulgidus TiaS-tRNA(Ile2) complexed with ATP, or with AMPCPP and agmatine, revealing a previously unknown kinase module required for activating C34 by phosphorylation, and showing the molecular mechanism by which TiaS discriminates between tRNA(Ile2) and tRNA(Met). In the TiaS-tRNA(Ile2)-ATP complex, C34 is trapped within a pocket far away from the ATP-binding site. In the agmatine-containing crystals, C34 is located near the AMPCPP gamma-phosphate in the kinase module, demonstrating that agmatine is essential for placing C34 in the active site. These observations also provide the structural dynamics for agm(2)C formation..
24. Yukimatsu Toh, Daijiro Takeshita, Takashi Nagaike, Tomoyuki Numata, Kozo Tomita, Mechanism for the Alteration of the Substrate Specificities of Template-Independent RNA Polymerases, STRUCTURE, 10.1016/j.str.2010.12.006, 19, 2, 232-243, 2011.02, PolyA polymerase (PAP) adds a polyA tail onto the 3'-end of RNAs without a nucleic acid template, using adenosine-5'-triphosphate (ATP) as a substrate. The mechanism for the substrate selection by eubacterial PAP remains obscure. Structural and biochemical studies of Escherichia coli PAP (EcPAP) revealed that the shape and size of the nucleobase-interacting pocket of EcPAP are maintained by an intra-molecular hydrogen-network, making it suitable for the accommodation of only ATP, using a single amino acid, Arg(197). The pocket structure is sustained by interactions between the catalytic domain and the RNA-binding domain. EcPAP has a flexible basic C-terminal region that contributes to optimal RNA translocation for processive adenosine 5'-monophosphate (AMP) incorporations onto the 3'-end of RNAs. A comparison of the EcPAP structure with those of other template-independent RNA polymerases suggests that structural changes of domain(s) outside the conserved catalytic core domain altered the substrate specificities of the template-independent RNA polymerases..
25. Takuo Osawa, Hideko Inanaga, Satoshi Kimura, Naohiro Terasaka, Tsutomu Suzuki, Tomoyuki Numata, Crystallization and preliminary X-ray diffraction analysis of an archaeal tRNA-modification enzyme, TiaS, complexed with tRNA(Ile2) and ATP, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 10.1107/S1744309111034890, 67, Pt 11, 1414-1416, 2011.11, The cytidine at the first anticodon position of archaeal tRNA(Ile2), which decodes the isoleucine AUA codon, is modified to 2-agmatinylcytidine (agm(2)C) to guarantee the fidelity of protein biosynthesis. This post-transcriptional modification is catalyzed by tRNA(Ile)-agm(2)C synthetase (TiaS) using ATP and agmatine as substrates. Archaeoglobus fulgidus TiaS was overexpressed in Escherichia coli cells and purified. tRNA(Ile2) was prepared by in vitro transcription with T7 RNA polymerase. TiaS was cocrystallized with both tRNA(Ile2) and ATP by the vapour-diffusion method. The crystals of the TiaS-tRNA(Ile2)-ATP complex diffracted to 2.9 angstrom resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the primitive hexagonal space group P3(2)21, with unit-cell parameters a = b = 131.1, c = 86.6 angstrom. The asymmetric unit is expected to contain one TiaS-tRNA(Ile2)-ATP complex, with a Matthews coefficient of 2.8 angstrom(3) Da(-1) and a solvent content of 61%..
26. Takayuki Ohnuma, Tomoyuki Numata, Takuo Osawa, Mamiko Mizuhara, Kjell M. Varum, Tamo Fukamizo, Crystal structure and mode of action of a class V chitinase from Nicotiana tabacum, PLANT MOLECULAR BIOLOGY, 10.1007/s11103-010-9727-z, 75, 3, 291-304, 2011.02, A class V chitinase from Nicotiana tabacum (NtChiV) with amino acid sequence similar to that of Serratia marcescens chitinase B (SmChiB) was expressed in E. coli and purified to homogeneity. When N-acetylglucosamine oligosaccharides [(NAG)(n)] were hydrolyzed by the purified NtChiV, the second glycosidic linkage from the non-reducing end was predominantly hydrolyzed in a manner similar to that of SmChiB. NtChiV was shown to hydrolyze partially N-acetylated chitosan non-processively, whereas SmChiB hydrolyzes the same substrate processively. The crystal structure of NtChiV was determined by the single-wavelength anomalous dispersion method at 1.2 resolution. The protein adopts a classical (beta/alpha)(8)-barrel fold (residues 1-233 and 303-348) with an insertion of a small (alpha + beta) domain (residues 234-302). This is the first crystal structure of a plant class V chitinase. The crystal structure of the inactive mutant NtChiV E115Q complexed with (NAG)(4) was also solved and exhibited a linear conformation of the bound oligosaccharide occupying -2, +1, +2, and +3 subsites. The complex structure corresponds to an initial state of (NAG)(4) binding, which is proposed to be converted into a bent conformation through sliding of the +1, +2, and +3 sugar units to -1, +1, and +2 subsites. Although NtChiV is similar to SmChiB, the chitin-binding domain is present in the C-terminus of the latter, but not in the former. Aromatic amino acid residues found in the substrate binding cleft of SmChiB, including Trp97, are substituted with aliphatic residues in NtChiV. These structural differences appear to be responsible for NtChiV being a non-processive enzyme..
27. Naohiro Terasaka, Satoshi Kimura, Takuo Osawa, Tomoyuki Numata, Tsutomu Suzuki, Biogenesis of 2-agmatinylcytidine catalyzed by the dual protein and RNA kinase TiaS, NATURE STRUCTURAL & MOLECULAR BIOLOGY, 10.1038/nsmb.2121, 18, 11, 1268-U116, 2011.11, The archaeal AUA-codon specific tRNA(Ile) contains 2-agmatinylcytidine (agm(2)C or agmatidine) at the anticodon wobble position (position 34). The formation of this essential modification is catalyzed by tRNA(Ile)-agm(2)C synthetase (TiaS) using agmatine and ATP as substrates. TiaS has a previously unknown catalytic domain, which we have named the Thr18-Cyt34 kinase domain (TCKD). Biochemical analyses of Archaeoglobus fulgidus TiaS and its mutants revealed that the TCKD first hydrolyzes ATP into AMP and pyrophosphate, then phosphorylates the C2 position of C34 with the gamma-phosphate. Next, the amino group of agmatine attacks this position to release the phosphate and form agm(2)C. Notably, the TCKD also autophosphorylates the Thr18 of TiaS, which may be involved in agm(2)C formation. Thus, the unique kinase domain of TiaS catalyzes dual phosphorylation of protein and RNA substrates..
28. Takayuki Ohnuma, Tomoyuki Numata, Takuo Osawa, Mamiko Mizuhara, Outi Lampela, Andre H. Juffer, Karen Skriver, Tamo Fukamizo, A class V chitinase from Arabidopsis thaliana: gene responses, enzymatic properties, and crystallographic analysis, PLANTA, 10.1007/s00425-011-1390-3, 234, 1, 123-137, 2011.07, Expression of a class V chitinase gene (At4g19810, AtChiC) in Arabidopsis thaliana was examined by quantitative real-time PCR and by analyzing microarray data available at Genevestigator. The gene expression was induced by the plant stress-related hormones abscisic acid (ABA) and jasmonic acid (JA) and by the stress resulting from the elicitor flagellin, NaCl, and osmosis. The recombinant AtChiC protein was produced in E. coli, purified, and characterized with respect to the structure and function. The recombinant AtChiC hydrolyzed N-acetylglucosamine oligomers producing dimers from the non-reducing end of the substrates. The crystal structure of AtChiC was determined by the molecular replacement method at 2.0 resolution. AtChiC was found to adopt an (beta/alpha)(8) fold with a small insertion domain composed of an alpha-helix and a five-stranded beta-sheet. From docking simulation of AtChiC with pentameric substrate, the amino acid residues responsible for substrate binding were found to be well conserved when compared with those of the class V chitinase from Nicotiana tabacum (NtChiV). All of the structural and functional properties of AtChiC are quite similar to those obtained for NtChiV, and seem to be common to class V chitinases from higher plants..
29. Naoyuki Umemoto, Takayuki Ohnuma, Henri Urpilainen, Takanori Yamamoto, Tomoyuki Numata, Tamo Fukamizo, Role of Tryptophan Residues in a Class V Chitinase from Nicotiana tabacum, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 10.1271/bbb.110914, 76, 4, 778-784, 2012.04, Tryptophan residues located in the substrate-binding cleft of a class V chitinase from Nicotiana tabacum (NtChiV) were mutated to alanine and phenylalanine (W190F, W326F, W190F/W326F, W190A, W326A, and W190A/W326A), and the mutant enzymes were characterized to define the role of the tryptophans. The mutations of Trp326 lowered thermal stability by 5-7 degrees C, while the mutations of Trp190 lowered stability only by 2-4 degrees C. The Trp326 mutations strongly impaired enzymatic activity, while the effects of the Trp190 mutations were moderate. The experimental data were rationalized based on the crystal structure of NtChiV in a complex with (GlcNAc)(4), in which Trp190 is exposed to the solvent and involved in face-to-face stacking interaction with the +2 sugar, while Trp326 is buried inside but interacts with the -2 sugar through hydrophobicity. HPLC analysis of anomers of the enzymatic products suggested that Trp190 specifically recognizes the beta-anomer of the +2 sugar. The strong effects of the Trp326 mutations on activity and stability suggest multiple roles of the residue in stabilizing the protein structure, in sugar residue binding at subsite -2, and probably in maintaining catalytic efficiency by providing a hydrophobic environment for proton donor Glu115..
30. Takayuki Ohnuma, Tomoyuki Numata, Takuo Osawa, Hideko Inanaga, Yoko Okazaki, Shoko Shinya, Kaori Kondo, Tatsuya Fukuda, Tamo Fukamizo, Crystal structure and chitin oligosaccharide-binding mode of a loopful' family GH19 chitinase from rye, Secale cereale, seeds, FEBS JOURNAL, 10.1111/j.1742-4658.2012.08723.x, 279, 19, 3639-3651, 2012.10, The substrate-binding mode of a 26-kDa GH19 chitinase from rye, Secale cereale, seeds (RSC-c) was investigated by crystallography, site-directed mutagenesis and NMR spectroscopy. The crystal structure of RSC-c in a complex with an N-acetylglucosamine tetramer, (GlcNAc)4, was successfully solved, and revealed the binding mode of the tetramer to be an aglycon-binding site, subsites +1, +2, +3, and +4. These are the first crystallographic data showing the oligosaccharide-binding mode of a family GH19 chitinase. From HPLC analysis of the enzymatic reaction products, mutation of Trp72 to alanine was found to affect the product distribution obtained from the substrate, p-nitrophenyl penta-N-acetyl-beta-chitopentaoside. Mutational experiments confirmed the crystallographic finding that the Trp72 side chain interacts with the +4 moiety of the bound substrate. To further confirm the crystallographic data, binding experiments were also conducted in solution using NMR spectroscopy. Several signals in the 1H15N HSQC spectrum of the stable isotope-labeled RSC-c were affected upon addition of (GlcNAc)4. Signal assignments revealed that most signals responsive to the addition of (GlcNAc)4 are derived from amino acids located at the surface of the aglycon-binding site. The binding mode deduced from NMR binding experiments in solution was consistent with that from the crystal structure. Database ?The atomic coordinates and structural factors have been deposited in the Protein Data Bank, under the accession codes 4DWX (unliganded form) and 4DYG ((GlcNAc)4 complex). Chitinase, EC 3.2.1.14. Backbone assignment data were deposited in the Biological Magnetic Resonance Data Bank ( http://www.bmrb.wisc.edu/bmrb/) with the code number 11467 Structured digital abstract RSC-c and RSC-c bind by x-ray crystallography (View interaction).
31. Takuo Osawa, Hideko Inanaga, Tomoyuki Numata, Crystallization and preliminary X-ray diffraction analysis of the Cmr2-Cmr3 subcomplex in the CRISPR-Cas RNA-silencing effector complex, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 10.1107/S1744309113011202, 69, Pt 5, 585-587, 2013.05, Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, found in prokaryotes, are transcribed to produce CRISPR RNAs (crRNAs). The Cmr proteins (Cmr1-6) and crRNA form a ribonucleoprotein complex that degrades target RNAs derived from invading genetic elements. Cmr2dHD, a Cmr2 variant lacking the N-terminal putative HD nuclease domain, and Cmr3 were co-expressed in Escherichia coli cells and co-purified as a complex. The Cmr2dHD-Cmr3 complex was co-crystallized with 3'-AMP by the vapour-diffusion method. The crystals diffracted to 2.6 angstrom resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 103.9, b = 136.7, c = 192.0 angstrom. The asymmetric unit of the crystals is expected to contain one Cmr2dHD-Cmr3 complex with a Matthews coefficient of 3.0 angstrom (3) Da(-1) and a solvent content of 59%..
32. Takayuki Ohnuma, Naoyuki Umemoto, Toki Taira, Tamo Fukamizo, Tomoyuki Numata, Crystallization and preliminary X-ray diffraction analysis of an active-site mutant of 'loopless' family GH19 chitinase from Bryum coronatum in a complex with chitotetraose, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 10.1107/S1744309113028935, 69, Pt 12, 1360-1362, 2013.12, The catalytic mechanism of family GH19 chitinases is not well understood owing to insufficient information regarding the three-dimensional structures of enzyme-substrate complexes. Here, the crystallization and preliminary X-ray diffraction analysis of a selenomethionine-labelled active-site mutant of 'loopless' family GH19 chitinase from the moss Bryum coronatum in complex with chitotetraose, (GlcNAc) 4, are reported. The crystals were grown using the vapour-diffusion method. They diffracted to 1.58 angstrom resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 74.5, b = 58.4, c = 48.1 angstrom, beta = 115.6 degrees. The asymmetric unit of the crystals is expected to contain one protein molecule, with a Matthews coefficient of 2.08 angstrom(3) Da(-1) and a solvent content of 41%..
33. Takuo Osawa, Hideko Inanaga, Tomoyuki Numata, Crystal Structure of the Cmr2-Cmr3 Subcomplex in the CRISPR-Cas RNA Silencing Effector Complex, JOURNAL OF MOLECULAR BIOLOGY, 10.1016/j.jmb.2013.03.042, 425, 20, 3811-3823, 2013.10, Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci found in prokaryotes are transcribed to produce CRISPR RNAs (crRNAs) that, together with CRISPR-associated (Cas) proteins, target and degrade invading genetic materials. Cmr proteins (Cmr1-6) and crRNA form a sequence-specific RNA silencing effector complex. Here, we report the crystal structures of the Pyrococcus furiosus Cmr2-Cmr3 subcomplex bound with nucleotides (3'-AMP or ATP). The association of Cmr2 and Cmr3 forms an idiosyncratic crevasse, which binds the nucleotides. Cmr3 shares structural similarity with Cas6, which cleaves precursor crRNA for maturation, suggesting the divergent evolution of these proteins. Due to the structural resemblance, the properties of the RNA binding surface observed in Cas6 are well conserved in Cmr3, indicating the RNA binding ability of Cmr3. This surface of Cmr3 constitutes the crevasse observed in the Cmr2-Cmr3 complex. Our findings suggest that the Cmr2-Cmr3 complex uses the crevasse to bind crRNA and/or substrate RNA during the reaction. (C) 2013 Elsevier Ltd. All rights reserved..
34. Takayuki Ohnuma, Naoyuki Umemoto, Kaori Kondo, Tomoyuki Numata, Tamo Fukamizo, Complete subsite mapping of a "loopful" GH19 chitinase from rye seeds based on its crystal structure, FEBS LETTERS, 10.1016/j.febslet.2013.07.008, 587, 16, 2691-2697, 2013.08, Crystallographic analysis of a mutated form of "loopful" GH19 chitinase from rye seeds a double mutant RSC-c, in which Glu67 and Trp72 are mutated to glutamine and alanine, respectively, (RSC-c-E67Q/W72A) in complex with chitin tetrasaccharide (GlcNAc)(4) revealed that the entire substrate-binding cleft was completely occupied with the sugar residues of two (GlcNAc)(4) molecules. One (GlcNAc)(4) molecule bound to subsites -4 to -1, while the other bound to subsites +1 to +4. Comparisons of the main chain conformation between liganded RSC-c-E67Q/W72A and unliganded wild type RSC-c suggested domain motion essential for catalysis. This is the first report on the complete subsite mapping of GH19 chitinase. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved..
35. Ogata M, Umemoto N, Ohnuma T, Numata T, Suzuki A, Usui T, Fukamizo T, A novel transition-state analogue for lysozyme, 4-O-β-tri-N-acetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate., The Journal of biological chemistry, 10.1074/jbc.M112.439281, 288, 9, 6072-6082, 2013.03.
36. Takayuki Ohnuma, Naoyuki Umemoto, Takuya Nagata, Shoko Shinya, Tomoyuki Numata, Toki Taira, Tamo Fukamizo, Crystal structure of a "loopless" GH19 chitinase in complex with chitin tetrasaccharide spanning the catalytic center, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 10.1016/j.bbapap.2014.02.013, 1844, 4, 793-802, 2014.04, Descriptions: The structure of a GH19 chitinase from the moss Bryum coronatum (BcChi-A) in complex with the substrate was examined by X-ray crystallography and NMR spectroscopy in solution. The X-ray crystal structure of the inactive mutant of BcChi-A (BcChi-A-E61A) liganded with chitin tetramer (GlcNAc)(4) revealed a clear electron density of the tetramer bound to subsites -2, -1, +1, and +2. Individual sugar residues were recognized by several amino acids at these subsites through a number of hydrogen bonds. This is the first crystal structure of GH19 chitinase liganded with oligosaccharide spanning the catalytic center. NMR titration experiments of chitin oligosaccharides into the BcChi-A-E61A solution showed that the binding mode observed in the crystal structure is similar to that in solution. The C-1 carbon of -1 GlcNAc, the O epsilon 1 atom of the catalytic base (Glu70), and the O gamma atom of Ser102 form a "triangle" surrounding the catalytic water, and the arrangement structurally validated the proposed catalytic mechanism of GH19 chitinases. The glycosidic linkage between -1 and +1 sugars was found to be twisted and under strain. This situation may contribute to the reduction of activation energy for hydrolysis. The complex structure revealed a more refined mechanism of the chitinase catalysis. (C) 2014 Elsevier B.V. All rights reserved..
37. Tsutomu Suzuki, Tomoyuki Numata, Convergent evolution of AUA decoding in bacteria and archaea, RNA BIOLOGY, 10.4161/15476286.2014.992281, 11, 12, 1586-1596, 2014.12, Deciphering AUA codons is a difficult task for organisms, because AUA and AUG specify isoleucine (Ile) and methionine (Met), separately. Each of the other purine-ending sense co-don sets (NNR) specifies a single amino acid in the universal genetic code. In bacteria and archaea, the cytidine derivatives, 2-lysylcytidine (L or lysidine) and 2-agmatinylcytidine (agm(2)C or agmatidine), respectively, are found at the first letter of the anticodon of tRNA(Ile) responsible for AUA codons. These modifications prevent base pairing with G of the third letter of AUG codon, and enable tRNA(Ile) to decipher AUA codon specifically. In addition, these modifications confer a charging ability of tRNA(Ile) with Ile. Despite their similar chemical structures, L and agm(2)C are synthesized by distinctive mechanisms and catalyzed by different classes of enzymes, implying that the analogous decoding systems for AUA codons were established by convergent evolution after the phylogenic split between bacteria and archaea-eukaryotes lineages following divergence from the last universal common ancestor (LUCA)..
38. Naoyuki Umemoto, Takayuki Ohnuma, Takuo Osawa, Tomoyuki Numata, Tamo Fukamizo, Modulation of the transglycosylation activity of plant family GH18 chitinase by removing or introducing a tryptophan side chain, FEBS LETTERS, 10.1016/j.febslet.2015.07.018, 589, 18, 2327-2333, 2015.08, Transglycosylation (TG) activity of a family GH18 chitinase from the cycad, Cycas revoluta, (CrChiA) was modulated by removing or introducing a tryptophan side chain. The removal from subsite +3 through mutation of Trp168 to alanine suppressed TG activity, while introduction into subsite +1 through mutation of Gly77 to tryptophan (CrChiA-G77W) enhanced TG activity. The crystal structures of an inactive double mutant of CrChiA (CrChiA-G77W/E119Q) with one or two N-acetylglucosamine residues occupying subsites +1 or +1/+2, respectively, revealed that the Trp77 side chain was oriented toward +1 GlcNAc to be stacked with it face-to-face, but rotated away from subsite +1 in the absence of GlcNAc at the subsite. Aromatic residues in the aglycon-binding site are key determinants of TG activity of GH18 chitinases. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved..
39. Tomoyuki Numata, Mechanisms of the tRNA wobble cytidine modification essential for AUA codon decoding in prokaryotes, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 10.1080/09168451.2014.975185, 79, 3, 347-353, 2015.03, Bacteria and archaea have 2-lysylcytidine (L or lysidine) and 2-agmatinylcytidine (agm(2)C or agmatidine), respectively, at the first (wobble) position of the anticodon of the AUA codon-specific tRNA(Ile). These lysine- or agmatine-conjugated cytidine derivatives are crucial for the precise decoding of the genetic code. L is synthesized by tRNA(Ile)-lysidine synthetase (TilS), which uses l-lysine and ATP as substrates. Agm(2)C formation is catalyzed by tRNA(Ile)-agm(2)C synthetase (TiaS), which uses agmatine and ATP for the reaction. Despite the fact that TilS and TiaS synthesize structurally similar cytidine derivatives, these enzymes belong to non-related protein families. Therefore, these enzymes modify the wobble cytidine by distinct catalytic mechanisms, in which TilS activates the C2 carbon of the wobble cytidine by adenylation, while TiaS activates it by phosphorylation. In contrast, TilS and TiaS share similar tRNA recognition mechanisms, in which the enzymes recognize the tRNA acceptor stem to discriminate tRNA(Ile) and tRNA(Met)..
40. Takuo Osawa, Hideko Inanaga, Tomoyuki Numata, Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex, Acta Crystallographica Section F:Structural Biology Communications, 10.1107/S2053230X15007104, 71, 735-740, 2015.06, Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR-Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR-Cas Cmr complex, composed of the six Cas proteins (Cmr1-Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (CmrΔ1) complex composed of Pyrococcus furiosus Cmr2-Cmr3, Archaeoglobus fulgidus Cmr4-Cmr5-Cmr6 and the 39-mer P. furiosus 7.01-crRNA was prepared. The CmrΔ1 complex was cocrystallized with single-stranded DNA (ssDNA) complementary to the crRNA guide by the vapour-diffusion method. The crystals diffracted to 2.1 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 75.5, b = 76.2, c = 139.2 Å, α = 90.3, β = 104.8, γ = 118.6°. The asymmetric unit of the crystals is expected to contain one CmrΔ1-ssDNA complex, with a Matthews coefficient of 2.03 Å&lt
sup&gt
3&lt
/sup&gt
Da&lt
sup&gt
-1&lt
/sup&gt
and a solvent content of 39.5%..
41. Takuo Osawa, Hideko Inanaga, Tomoyuki Numata, Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, 10.1107/S2053230X15007104, 71, Pt 6, 735-740, 2015.06, Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR-Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR-Cas Cmr complex, composed of the six Cas proteins (Cmr1-Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (Cmr Delta 1) complex composed of Pyrococcus furiosus Cmr2-Cmr3, Archaeoglobus fulgidus Cmr4-Cmr5-Cmr6 and the 39-mer P. furiosus 7.01-crRNA was prepared. The Cmr Delta 1 complex was cocrystallized with single-stranded DNA (ssDNA) complementary to the crRNA guide by the vapour-diffusion method. The crystals diffracted to 2.1 angstrom resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 75.5, b = 76.2, c = 139.2 angstrom, alpha = 90.3, beta = 104.8, gamma = 118.6 degrees. The asymmetric unit of the crystals is expected to contain one Cmr Delta 1-ssDNA complex, with a Matthews coefficient of 2.03 angstrom(3) Da(-1) and a solvent content of 39.5%..
42. Naoyuki Umemoto, Yuka Kanda, Takayuki Ohnuma, Takuo Osawa, Tomoyuki Numata, Shohei Sakuda, Toki Taira, Tamo Fukamizo, Crystal structures and inhibitor binding properties of plant class V chitinases: the cycad enzyme exhibits unique structural and functional features, PLANT JOURNAL, 10.1111/tpj.12785, 82, 1, 54-66, 2015.04, A classV (glycoside hydrolase family18) chitinase from the cycad Cycas revoluta (CrChiA) is a plant chitinase that has been reported to possess efficient transglycosylation (TG) activity. We solved the crystal structure of CrChiA, and compared it with those of classV chitinases from Nicotiana tabacum (NtChiV) and Arabidopsis thaliana (AtChiC), which do not efficiently catalyze the TG reaction. All three chitinases had a similar (/)(8) barrel fold with an (+) insertion domain. In the acceptor binding site (+1, +2 and +3) of CrChiA, the Trp168 side chain was found to stack face-to-face with the +3 sugar. However, this interaction was not found in the identical regions of NtChiV and AtChiC. In the DxDxE motif, which is essential for catalysis, the carboxyl group of the middle Asp (Asp117) was always oriented toward the catalytic acid Glu119 in CrChiA, whereas the corresponding Asp in NtChiV and AtChiC was oriented toward the first Asp. These structural features of CrChiA appear to be responsible for the efficient TG activity. When binding of the inhibitor allosamidin was evaluated using isothermal titration calorimetry, the changes in binding free energy of the three chitinases were found to be similar to each other, i.e. between -9.5 and -9.8kcal mol(-1). However, solvation and conformational entropy changes in CrChiA were markedly different from those in NtChiV and AtChiC, but similar to those of chitinaseA from Serratia marcescens (SmChiA), which also exhibits significant TG activity. These results provide insight into the molecular mechanism underlying the TG reaction and the molecular evolution from bacterial chitinases to plant classV chitinases..
43. Tomoyuki Numata, Hideko Inanaga, Chikara Sato, Takuo Osawa, Crystal Structure of the Csm3-Csm4 Subcomplex in the Type III-A CRISPR-Cas Interference Complex, JOURNAL OF MOLECULAR BIOLOGY, 10.1016/j.jmb.2014.09.029, 427, 2, 259-273, 2015.01, Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci play a pivotal role in the prokaryotic host defense system against invading genetic materials. The CRISPR loci are transcribed to produce CRISPR RNAs (crRNAs), which form interference complexes with CRISPR-associated (Cas) proteins to target the invading nucleic acid for degradation. The interference complex of the type III-A CRISPR Cas system is composed of five Cas proteins (Csm1-Csm5) and a crRNA, and targets invading DNA. Here, we show that the Csm1, Csm3, and Csm4 proteins from Methanocaldococcus jannaschii form a stable subcomplex. We also report the crystal structure of the M. jannaschii Csm3 Csm4 subcomplex at 3.1 angstrom resolution. The complex structure revealed the presence of a basic concave surface around their interface, suggesting the RNA and/or DNA binding ability of the complex. A gel retardation analysis showed that the Csm3 Csm4 complex binds single-stranded RNA in a non-sequence-specific manner. Csm4 structurally resembles Cmr3, a component of the type III-B CRISPR-Cas interference complex. Based on bioinformatics, we constructed a model structure of the Csm1 Csm4 Csm3 ternary complex, which provides insights into its role in the Csm interference complex. (C) 2014 Elsevier Ltd. All rights reserved..
44. Takuo Osawa, Hideko Inanaga, Chikara Sato, Tomoyuki Numata, Crystal Structure of the CRISPR-Cas RNA Silencing Cmr Complex Bound to a Target Analog, MOLECULAR CELL, 10.1016/j.molcel.2015.03.018, 58, 3, 418-430, 2015.05, In prokaryotes, Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNAs (crRNAs), together with CRISPR-associated (Cas) proteins, capture and degrade invading genetic materials. In the type III-B CRISPR-Cas system, six Cas proteins (Cmr1-Cmr6) and a crRNA form an RNA silencing Cmr complex. Here we report the 2.1 angstrom crystal structure of the Cmr1-deficient, functional Cmr complex bound to single-stranded DNA, a substrate analog complementary to the crRNA guide. Cmr3 recognizes the crRNA 5' tag and defines the start position of the guide-target duplex, using its idiosyncratic loops. The beta-hairpins of three Cmr4 subunits intercalate within the duplex, causing nucleotide displacements with 6 nt intervals, and thus periodically placing the scissile bonds near the crucial aspartate of Cmr4. The structure reveals the mechanism for specifying the periodic target cleavage sites from the crRNA 5' tag and provides insights into the assembly of the type III interference machineries and the evolution of the Cmr and Cascade complexes..
45. Yoshihito Kitaoku, Naoyuki Umemoto, Takayuki Ohnuma, Tomoyuki Numata, Toki Taira, Shohei Sakuda, Tamo Fukamizo, A class III chitinase without disulfide bonds from the fern, Pteris ryukyuensis: crystal structure and ligand-binding studies, PLANTA, 10.1007/s00425-015-2330-4, 242, 4, 895-907, 2015.10, We first solved the crystal structure of class III catalytic domain of a chitinase from fern (PrChiA-cat), and found a structural difference between PrChiA-cat and hevamine. PrChiA-cat was found to have reduced affinities to chitin oligosaccharides and allosamidin.
Plant class III chitinases are subdivided into enzymes with three disulfide bonds and those without disulfide bonds. We here referred to the former enzymes as class IIIa chitinases and the latter as class IIIb chitinases. In this study, we solved the crystal structure of the class IIIb catalytic domain of a chitinase from the fern Pteris ryukyuensis (PrChiA-cat), and compared it with that of hevamine, a class IIIa chitinase from Hevea brasiliensis. PrChiA-cat was found to adopt an (alpha/beta)(8) fold typical of GH18 chitinases in a similar manner to that of hevamine. However, PrChiA-cat also had two large loops that extruded from the catalytic site, and the corresponding loops in hevamine were markedly smaller than those of PrChiA-cat. An HPLC analysis of the enzymatic products revealed that the mode of action of PrChiA-cat toward chitin oligosaccharides, (GlcNAc) (n) (n = 4-6), differed from those of hevamine and the other class IIIa chitinases. The binding affinities of (GlcNAc)(3) and (GlcNAc)(4) toward the inactive mutant of PrChiA-cat were determined by isothermal titration calorimetry, and were markedly lower than those toward other members of the GH18 family. The affinity and the inhibitory activity of allosamidin toward PrChiA-cat were also lower than those toward the GH18 chitinases investigated to date. Several hydrogen bonds found in the crystal structure of hevamine-allosamidin complex were missing in the modeled structure of PrChiA-cat-allosamidin complex. The structural findings for PrChiA-cat successfully interpreted the functional data presented..
46. Shoko Shinya, Shigenori Nishimura, Yoshihito Kitaoku, Tomoyuki Numata, Hisashi Kimoto, Hideo Kusaoke, Takayuki Ohnuma, Tamo Fukamizo, Mechanism of chitosan recognition by CBM32 carbohydrate-binding modules from a Paenibacillus sp IK-5 chitosanase/glucanase, BIOCHEMICAL JOURNAL, 10.1042/BCJ20160045, 473, 8, 1085-1095, 2016.04, An antifungal chitosanase/glucanase isolated from the soil bacterium Paenibacillus sp. IK-5 has two CBM32 chitosan-binding modules (DD1 and DD2) linked in tandem at the C-terminus. In order to obtain insights into the mechanism of chitosan recognition, the structures of DD1 and DD2 were solved by NMR spectroscopy and crystallography. DD1 and DD2 both adopted a beta-sandwich fold with several loops in solution as well as in crystals. On the basis of chemical shift perturbations in H-1-N-15-HSQC resonances, the chitosan tetramer (GlcN)(4) was found to bind to the loop region extruded from the core beta-sandwich of DD1 and DD2. The binding site defined by NMR in solution was consistent with the crystal structure of DD2 in complex with (GlcN)(3), in which the bound (GlcN)(3) stood upright on its non-reducing end at the binding site. Glu(14) of DD2 appeared to make an electrostatic interaction with the amino group of the non-reducing end GlcN, and Arg(31), Tyr(36) and Glu(61) formed several hydrogen bonds predominantly with the non-reducing end GlcN. No interaction was detected with the reducing end GlcN. Since Tyr(36) of DD2 is replaced by glutamic acid in DD1, the mutation of Tyr(36) to glutamic acid was conducted in DD2 (DD2-Y36E), and the reverse mutation was conducted in DD1 (DD1-E36Y). Ligand-binding experiments using the mutant proteins revealed that this substitution of the 36th amino acid differentiates the binding properties of DD1 and DD2, probably enhancing total affinity of the chitosanase/glucanase toward the fungal cell wall..
47. Takayuki Ohnuma, Toki Taira, Naoyuki Umemoto, Yoshihito Kitaoku, Morten Sorlie, Tomoyuki Numata, Tamo Fukamizo, Crystal structure and thermodynamic dissection of chitin oligosaccharide binding to the LysM module of chitinase-A from Pteris ryukyuensis, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2017.08.143, 494, 3-4, 736-741, 2017.12, We determined the crystal structure of a LysM module from Pteris ryukyuensis chitinase-A (PrLysM2) at a resolution of 1.8 angstrom. Structural and binding analysis of PrLysM2 indicated that this module recognizes chitin oligosaccharides in a shallow groove comprised of five sugar-binding subsites on one side of the molecule. The free energy changes (Delta G(r)degrees) for binding of (GIcNAc)(6), (G1cNAc)(5), and (GIcNAc)(4) to PrLysM2 were determined to be -5.4, -5,4 and -4.6 kcal mol(-1), respectively, by ITC. Thermodynamic dissection of the binding energetics of (GIcNAc)(6) revealed that the driving force is the enthalpy change (Delta H-r degrees = -11.7 +/- 0.2 kcal/mol) and the solvation entropy change (-T Delta S-solv degrees = -5.9 +/- 0.6 kcal/mol). This is the first description of thermodynamic signatures of a chitin oligosaccharide binding to a LysM module. (C) 2017 Elsevier Inc. All rights reserved..
48. Yoshihito Kitaoku, Tamo Fukamizo, Tomoyuki Numata, Takayuki Ohnuma, Chitin oligosaccharide binding to the lysin motif of a novel type of chitinase from the multicellular green alga, Volvox carteri, PLANT MOLECULAR BIOLOGY, 10.1007/s11103-016-0549-5, 93, 1-2, 97-108, 2017.01, The chitinase-mediated defense system in higher plants has been intensively studied from physiological and structural viewpoints. However, the defense system in the most primitive plant species, such as green algae, has not yet been elucidated in details. In this study, we solved the crystal structure of a family CBM-50 LysM module attached to the N-terminus of chitinase from Volvox carteri, and successfully analyzed its chitin-binding ability by NMR spectroscopy and isothermal titration calorimetry. Trp96 of the LysM module appeared to make a CH-pi stacking interaction with the reducing end sugar residue of the ligand. We believe the data included in this manuscript provide novel insights into the molecular basis of chitinase-mediated defense system in green algae.
A chitinase from the multicellular green alga, Volvox carteri, contains two N-terminal lysin motifs (VcLysM1 and VcLysM2), that belong to the CBM-50 family, in addition to a catalytic domain. We produced a recombinant protein of VcLysM2 in order to examine its structure and function. The X-ray crystal structure of VcLysM2 was successfully solved at a resolution of 1.2 , and revealed that the protein adopts the beta alpha alpha beta fold typical of members belonging to the CBM-50 family. NMR spectra of C-13- and N-15-labeled proteins were analyzed in order to completely assign the main chain resonances of the H-1,N-15-HSQC spectrum in a sequential manner. NMR-based titration experiments of chitin oligosaccharides, (GlcNAc)(n) (n = 3-6), revealed the ligand-binding site of VcLysM2, in which the Trp96 side chain appeared to interact with the terminal GlcNAc residue of the ligand. We then mutated Trp96 to alanine (VcLysM2-W96A), and the mutant protein was characterized. Based on isothermal titration calorimetry, the affinity of (GlcNAc)(6) toward VcLysM2 (-6.9 kcal/mol) was found to be markedly higher than that of (GlcNAc)(3) (-4.1 kcal/mol), whereas the difference in affinities between (GlcNAc)(6) and (GlcNAc)(3) in VcLysM2-W96A (-5.1 and -4.0 kcal/mol, respectively) was only moderate. This suggests that the Trp96 side chain of VcLysM2 interacts with the sugar residue of (GlcNAc)(6) not with (GlcNAc)(3). VcLysM2 appears to preferentially bind (GlcNAc)(n) with longer chains and plays a major role in the degradation of the chitinous components of enzyme targets..
49. Tomoya Takashima, Tomoyuki Numata, Toki Taira, Tamo Fukamizo, Takayuki Ohnuma, Structure and Enzymatic Properties of a Two-Domain Family GH19 Chitinase from Japanese Cedar (Cryptomeria japonica) Pollen, Journal of Agricultural and Food Chemistry, 10.1021/acs.jafc.8b01140, 66, 22, 5699-5706, 2018.06, CJP-4 is an allergen found in pollen of the Japanese cedar Cryptomeria japonica. The protein is a two-domain family GH19 (class IV) Chitinase consisting of an N-terminal CBM18 domain and a GH19 catalytic domain. Here, we produced recombinant CJP-4 and CBM18-truncated CJP-4 (CJP-4-Cat) proteins. In addition to solving the crystal structure of CJP-4-Cat by X-ray crystallography, we analyzed the ability of both proteins to hydrolyze chitin oligosaccharides, (GlcNAc)n, polysaccharide substrates, glycol chitin, and β-chitin nanofiber and examined their inhibitory activity toward fungal growth. Truncation of the CBM18 domain did not significantly affect the mode of (GlcNAc)n hydrolysis. However, significant effects were observed when we used the polysaccharide substrates. The activity of CJP-4 toward the soluble substrate, glycol chitin, was lower than that of CJP-4-Cat. In contrast, CJP-4 exhibited higher activity toward β-chitin nanofiber, an insoluble substrate, than did CJP-4-Cat. Fungal growth was strongly inhibited by CJP-4 but not by CJP-4-Cat. These results indicate that the CBM18 domain assists the hydrolysis of insoluble substrate and the antifungal action of CJP-4-Cat by binding to chitin. CJP-4-Cat was found to have only two loops (loops I and III), as reported for ChiA, an allergenic class IV Chitinase from maize..
50. Synthetic ligands for PreQ1 riboswitches provide structural and mechanistic insights into targeting RNA tertiary structure..
51. Crystal structure and biochemical characterization of CJP38, a β-1,3-glucanase and allergen of Cryptomeria japonica pollen..
52. Takeshita D, Sato M, Inanaga H, Numata T, Crystal Structures of Csm2 and Csm3 in the Type III-A CRISPR-Cas Effector Complex., Journal of molecular biology, 10.1016/j.jmb.2019.01.009, 431, 4, 748-763, 2019.02.
53. Takamasa Teramoto, Takeshi Koyasu, Naruhiko Adachi, Masato Kawasaki, Toshio Moriya, Tomoyuki Numata, Toshiya Senda, Yoshimitsu Kakuta, Minimal protein-only RNase P structure reveals insights into tRNA precursor recognition and catalysis., The Journal of biological chemistry, 10.1016/j.jbc.2021.101028, 297, 3, 101028-101028, 2021.07, Ribonuclease P (RNase P) is an endoribonuclease that catalyzes the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). Ribonucleoprotein RNase P and protein-only RNase P (PRORP) in eukaryotes have been extensively studied, but the mechanism by which a prokaryotic nuclease recognizes and cleaves pre-tRNA is unclear. To gain insights into this mechanism, we studied homologs of Aquifex RNase P (HARPs), thought to be enzymes of approximately 23 kDa comprising only this nuclease domain. We determined the cryo-EM structure of Aq880, the first identified HARP enzyme. The structure unexpectedly revealed that Aq880 consists of both the nuclease and protruding helical (PrH) domains. Aq880 monomers assemble into a dimer via the PrH domain. Six dimers form a dodecamer with a left-handed one-turn superhelical structure. The structure also revealed that the active site of Aq880 is analogous to that of eukaryotic PRORPs. The pre-tRNA docking model demonstrated that 5' processing of pre-tRNAs is achieved by two adjacent dimers within the dodecamer. One dimer is responsible for catalysis, and the PrH domains of the other dimer are responsible for pre-tRNA elbow recognition. Our study suggests that HARPs measure an invariant distance from the pre-tRNA elbow to cleave the 5' leader sequence, which is analogous to the mechanism of eukaryotic PRORPs and the ribonucleoprotein RNase P. Collectively, these findings shed light on how different types of RNase P enzymes utilize the same pre-tRNA processing..
54. Daiki Kawamoto, Tomoya Takashima, Tamo Fukamizo, Tomoyuki Numata, Takayuki Ohnuma, A conserved loop structure of GH19 chitinases assists the enzyme function from behind the core-functional region., Glycobiology, 10.1093/glycob/cwab117, 2021.11, Plant GH19 chitinases have several loop structures, which may define their enzymatic properties. Among these loops, the longest loop, Loop-III, is most frequently conserved in GH19 enzymes. A GH19 chitinase from the moss Bryum coronatum (BcChi-A) has only one loop structure, Loop-III, which is connected to the catalytically important β-sheet region. Here, we produced and characterized a Loop-III-deleted mutant of BcChi-A (BcChi-A-ΔIII) and found that its stability and chitinase activity were strongly reduced. The deletion of Loop-III also moderately affected the chitooligosaccharide binding ability as well as the binding mode to the substrate-binding groove. The crystal structure of an inactive mutant of BcChi-A-ΔIII was successfully solved, revealing that the remaining polypeptide chain has an almost identical fold to that of the original protein. Loop-III is not necessarily essential for the folding of the enzyme protein. However, closer examination of the crystal structure revealed that the deletion of Loop-III altered the arrangement of the catalytic triad, Glu61, Glu70 and Ser102, and the orientation of the Trp103 side chain, which is important for sugar residue binding. We concluded that Loop-III is not directly involved in the enzymatic activity but assists the enzyme function by stabilizing the conformation of the β-sheet region and the adjacent substrate-binding platform from behind the core-functional regions..
55. Sumirtha Balaratnam, Curran Rhodes, Desta Doro Bume, Colleen Connelly, Christopher C Lai, James A Kelley, Kamyar Yazdani, Philip J Homan, Danny Incarnato, Tomoyuki Numata, John S Schneekloth Jr, A chemical probe based on the PreQ1 metabolite enables transcriptome-wide mapping of binding sites., Nature communications, 10.1038/s41467-021-25973-x, 12, 1, 5856-5856, 2021.10, The role of metabolite-responsive riboswitches in regulating gene expression in bacteria is well known and makes them useful systems for the study of RNA-small molecule interactions. Here, we study the PreQ1 riboswitch system, assessing sixteen diverse PreQ1-derived probes for their ability to selectively modify the class-I PreQ1 riboswitch aptamer covalently. For the most active probe (11), a diazirine-based photocrosslinking analog of PreQ1, X-ray crystallography and gel-based competition assays demonstrated the mode of binding of the ligand to the aptamer, and functional assays demonstrated that the probe retains activity against the full riboswitch. Transcriptome-wide mapping using Chem-CLIP revealed a highly selective interaction between the bacterial aptamer and the probe. In addition, a small number of RNA targets in endogenous human transcripts were found to bind specifically to 11, providing evidence for candidate PreQ1 aptamers in human RNA. This work demonstrates a stark influence of linker chemistry and structure on the ability of molecules to crosslink RNA, reveals that the PreQ1 aptamer/ligand pair are broadly useful for chemical biology applications, and provides insights into how PreQ1, which is similar in structure to guanine, interacts with human RNAs..
56. Yoshihito Kitaoku, Toki Taira, Tomoyuki Numata, Takayuki Ohnuma, Tamo Fukamizo, Structure, mechanism, and phylogeny of LysM-chitinase conjugates specifically found in fern plants., Plant science : an international journal of experimental plant biology, 10.1016/j.plantsci.2022.111310, 321, 111310-111310, 2022.08, A unique GH18 chitinase containing two N-terminal lysin motifs (PrLysM1 and PrLysM2) was first found in fern, Pteris ryukyuensis (Onaga and Taira, Glycobiology, 18, 414-423, 2008). This type of LysM-chitinase conjugates is not usually found in plants but in fungi. Here, we produced a similar GH18 chitinase with one N-terminal LysM module (EaLysM) from the fern, Equisetum arvense (EaChiA, Inamine et al., Biosci. Biotechnol. Biochem., 79, 1296-1304, 2015), using an Escherichia coli expression system and characterized for its structure and mechanism of action. The crystal structure of EaLysM exhibited an almost identical fold (βααβ) to that of PrLysM2. From isothermal titration calorimetry and nuclear magnetic resonance, the binding mode and affinities of EaLysM for chitooligosaccharides (GlcNAc)n (3, 4, 5, and 6) were found to be comparable to those of PrLysM2. The LysM module in EaChiA is likely to bind (GlcNAc)n almost independently through CH-π stacking of a Tyr residue with the pyranose ring. The (GlcNAc)n-binding mode of LysMs in the LysM-chitinase conjugates from fern plants appears to differ from that of plant LysMs acting in chitin- or Nod-signal perception, in which multiple LysMs cooperatively act on (GlcNAc)n. Phylogenetic analysis suggested that LysM-GH18 conjugates of fern plants formed a monophyletic group and had been separated earlier than forming the clade of fungal chitinases with LysMs..
57. Shunsuke Matsumoto, Suzuka Ono, Saori Shinoda, Chika Kakuta, Satoshi Okada, Takashi Ito, Tomoyuki Numata, Toshiya Endo, GET pathway mediates transfer of mislocalized tail-anchored proteins from mitochondria to the ER., The Journal of cell biology, 10.1083/jcb.202104076, 221, 6, 2022.06, Tail-anchored (TA) membrane proteins have a potential risk to be mistargeted to the mitochondrial outer membrane (OM). Such mislocalized TA proteins can be extracted by the mitochondrial AAA-ATPase Msp1 from the OM and transferred to the ER for ER protein quality control involving ubiquitination by the ER-resident Doa10 complex. Yet it remains unclear how the extracted TA proteins can move to the ER crossing the aqueous cytosol and whether this transfer to the ER is essential for the clearance of mislocalized TA proteins. Here we show by time-lapse microscopy that mislocalized TA proteins, including an authentic ER-TA protein, indeed move from mitochondria to the ER in a manner strictly dependent on Msp1 expression. The Msp1-dependent mitochondria-to-ER transfer of TA proteins is blocked by defects in the GET system, and this block is not due to impaired Doa10 functions. Thus, the GET pathway facilitates the transfer of mislocalized TA proteins from mitochondria to the ER..
58. Keisuke Oki, Mariko Nagata, Takeshi Yamagami, Tomoyuki Numata, Sonoko Ishino, Takuji Oyama, Yoshizumi Ishino, Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome., Nucleic acids research, 10.1093/nar/gkab799, 50, 7, 3601-3615, 2022.04, Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN-MCM-GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD's DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N-Gins51C-GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork..
59. Jun Tanaka, Tomoya Takashima, Naojiro Abe, Tamo Fukamizo, Tomoyuki Numata, Takayuki Ohnuma, Characterization of two rice GH18 chitinases belonging to family 8 of plant pathogenesis-related proteins., Plant science : an international journal of experimental plant biology, 10.1016/j.plantsci.2022.111524, 111524-111524, 2022.10, Two rice GH18 chitinases, Oschib1 and Oschib2, belonging to family 8 of plant pathogenesis-related proteins (PR proteins) were expressed, purified, and characterized. These enzymes, which have the structural features of class IIIb chitinases, preferentially cleaved the second glycosidic linkage from the non-reducing end of substrate chitin oligosaccharides as opposed to rice class IIIa enzymes, OsChib3a and OsChib3b, which mainly cleaved the fourth linkage from the non-reducing end of chitin hexasaccharide [(GlcNAc)6]. Oschib1 and Oschiab2 inhibited the growth of Fusarium solani, but showed only a weak or no antifungal activity against Aspergillus niger and Trichoderma viride on the agar plates. Structural analysis of Oschib1 and Oschib2 revealed that these enzymes have two large loops extruded from the (β/α)8 TIM-barrel fold, which are absent in the structures of class IIIa chitinases. The differences in the cleavage site preferences toward chitin oligosaccharides between plant class IIIa and IIIb chitinases are likely attributed to the additional loop structures found in the IIIb enzymes. The class IIIb chitinases, Oschib1 and Oschib2, seem to play important roles for the effective hydrolysis of chitin oligosaccharides released from the cell wall of the pathogenic fungi by the cooperative actions with the extracellular chitinases in rice..


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