記述言語：英語  

DOI： 10.1093/pnasnexus/pgae482

PubMed

担当区分：筆頭著者,　責任著者   記述言語：英語   出版者・発行元：Biochemical and Biophysical Research Communications  

Microorganisms synthesize a plethora of complex secondary metabolites, many of which are beneficial to human health, such as anticancer agents and antibiotics. Among these, the Sungeidines are a distinct class of secondary metabolites known for their bulky and intricate structures. They are produced by a specific biosynthetic gene cluster within the genome of the soil-dwelling actinomycete Micromonospora sp. MD118. A notable enzyme in the Sungeidine biosynthetic pathway is the activating sulfotransferase SgdX2. In this pathway, SgdX2 mediates a key sulfation step, after which the product undergoes spontaneous dehydration to yield a Sungeidine compound. To delineate the structural basis for SgdX2's substrate recognition and catalytic action, we have determined the crystal structure of SgdX2 in complex with its sulfate donor product, 3′-phosphoadenosine 5′-phosphate (PAP), at a resolution of 1.6 Å. Although SgdX2 presents a compact overall structure, its core elements are conserved among other activating sulfotransferases. Our structural analysis reveals a unique substrate-binding pocket that accommodates bulky, complex substrates, suggesting a specialized adaptation for Sungeidine synthesis. Moreover, we have constructed a substrate docking model that provides insights into the molecular interactions between SgdX2 and Sungeidine F, enhancing our understanding of the enzyme's specificity and catalytic mechanism. The model supports a general acid-base catalysis mechanism, akin to other sulfotransferases, and underscores the minor role of disordered regions in substrate recognition. This integrative study of crystallography and computational modeling advances our knowledge of microbial secondary metabolite biosynthesis and may facilitate the development of novel biotechnological applications.

DOI： 10.1016/j.bbrc.2024.149891

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PubMed

担当区分：筆頭著者   記述言語：英語   出版者・発行元：Glycobiology  

In various organisms, α1,3/α1,4-fucosyltransferases (CAZy GT10 family enzymes) mediate the assembly of type I (Galβ1,3GlcNAc) and/or type II (Galβ1,4GlcNAc)-based Lewis structures that are widely distributed in glycoconjugates. Unlike enzymes of other species, plant orthologues show little fucosyltransferase activity for type II-based glycans and predominantly catalyze the assembly of the Lewis A structure [Galβ1,3(Fucα1,4)GlcNAc] on the type I disaccharide unit of their substrates. However, the structural basis underlying this unique substrate selectivity remains elusive. In this study, we investigated the structure–function relationship of MiFUT13A, a mango α1,3/α1,4-fucosyltransferase. The prepared MiFUT13A displayed distinct α1,4-fucosyltransferase activity. Consistent with the enzymatic properties of this molecule, X-ray crystallography revealed that this enzyme has a typical GT-B fold-type structure containing a set of residues that are responsible for its SN2-like catalysis. Site-directed mutagenesis and molecular docking analyses proposed a rational binding mechanism for type I oligosaccharides. Within the catalytic cleft, the pocket surrounding Trp121 serves as a binding site, anchoring the non-reducing terminal β1,3-galactose that belongs to the type I disaccharide unit. Furthermore, Glu177 was postulated to function as a general base catalyst through its interaction with the 4-hydroxy group of the acceptor N-acetylglucosamine residue. Adjacent residues, specifically Thr120, Thr157 and Asp175 were speculated to assist in binding of the reducing terminal residues. Intriguingly, these structural elements were not fully conserved in mammalian orthologue which also shows predominant α1,4-fucosyltransferase activity. In conclusion, we have proposed that MiFUT13A generates the Lewis A structure on type I glycans through a distinct mechanism, divergent from that of mammalian enzymes.

DOI： 10.1093/glycob/cwae015

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PubMed

記述言語：英語   出版者・発行元：Journal of Biological Chemistry  

Ticks pose a substantial public health risk as they transmit various pathogens. This concern is related to the adept blood-sucking strategy of ticks, underscored by the action of the anticoagulant, madanin, which is known to exhibit an approximately 1000-fold increase in anticoagulant activity following sulfation of its two tyrosine residues, Tyr51 and Tyr54. Despite this knowledge, the molecular mechanism underlying sulfation by tick tyrosylprotein sulfotransferase (TPST) remains unclear. In this study, we successfully prepared tick TPST as a soluble recombinant enzyme. We clarified the method by which this enzyme proficiently sulfates tyrosine residues in madanin. Biochemical analysis using a substrate peptide based on madanin and tick TPST, along with the analysis of the crystal structure of the complex and docking simulations, revealed a sequential sulfation process. Initial sulfation at the Tyr51 site augments binding, thereby facilitating efficient sulfation at Tyr54. Beyond direct biochemical implications, these findings considerably improve our understanding of tick blood-sucking strategies. Furthermore, combined with the utility of modified tick TPST, our findings may lead to the development of novel anticoagulants, promising avenues for thrombotic disease intervention and advancements in the field of public health.

DOI： 10.1016/j.jbc.2024.105748

Scopus

PubMed

記述言語：英語   出版者・発行元：Journal of Bioscience and Bioengineering  

C-phycocyanin (CPC), which contains open-chain tetrapyrroles, is a major light-harvesting red-fluorescent protein with an important role in aquatic photosynthesis. Recently, we reported a non-conventional CPC from Thermoleptolyngbya sp. O-77 (CPCO77) that contains two different structures, i.e., a hexameric structure and a non-conventional octameric structure. However, the assembly and disassembly mechanisms of the non-conventional octameric form of CPC remain unclear. To understand this assembly mechanism, we performed an in vitro experiment to study the disassembly and reassembly behaviors of CPC using isolated CPC subunits. The dissociation of the CPCO77 subunit was performed using a Phenyl-Sepharose column in 20 mM potassium phosphate buffer (pH 6.0) containing 7.0 M urea. For the first time, crystals of isolated CPC subunits were obtained and analyzed after separation. After the removal of urea from the purified α and β subunits, we performed an in vitro reassembly experiment for CPC and analyzed the reconstructed CPC using spectrophotometric and X-ray crystal structure analyses. The crystal structure of the reassembled CPC was nearly identical to that of the original CPCO77. The findings of this study indicate that the octameric CPCO77 is a naturally occurring form in the thermophilic cyanobacterium Thermoleptolyngbya sp. O-77.

DOI： 10.1016/j.jbiosc.2023.12.015

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PubMed

記述言語：英語   出版者・発行元：PNAS Nexus  

Cytosolic sulfotransferases (SULTs) are cytosolic enzymes that catalyze the transfer of sulfonate group to key endogenous compounds, altering the physiological functions of their substrates. SULT enzymes catalyze the O-sulfonation of hydroxy groups or N-sulfonation of amino groups of substrate compounds. In this study, we report the discovery of C-sulfonation of α,β-unsaturated carbonyl groups mediated by a new SULT enzyme, SULT7A1, and human SULT1C4. Enzymatic assays revealed that SULT7A1 is capable of transferring the sulfonate group from 3′-phosphoadenosine 5′-phosphosulfate to the α-carbon of α,β-unsaturated carbonyl-containing compounds, including cyclopentenone prostaglandins as representative endogenous substrates. Structural analyses of SULT7A1 suggest that the C-sulfonation reaction is catalyzed by a novel mechanism mediated by His and Cys residues in the active site. Ligand-activity assays demonstrated that sulfonated 15-deoxy prostaglandin J2 exhibits antagonist activity against the prostaglandin receptor EP2 and the prostacyclin receptor IP. Modification of α,β-unsaturated carbonyl groups via the new prostaglandin-sulfonating enzyme, SULT7A1, may regulate the physiological function of prostaglandins in the gut. Discovery of C-sulfonation of α,β-unsaturated carbonyl groups will broaden the spectrum of potential substrates and physiological functions of SULTs.

DOI： 10.1093/pnasnexus/pgae097

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PubMed

出版者・発行元：Sensors and Materials  

Human orthogonal enzymes (HOEs) do not show the same activities as the endogenous enzymes of human cells and thus are useful as amplification enzymes to detect antigen proteins in biological samples. Here, we evaluate a new HOE from Escherichia coli, α-sulfoquinovosidase (α-SQase). We confirmed that the activity of α-SQase did not exist in examined human cell lines, and thus it was applicable to live-cell enzyme-linked immunosorbent assay (ELISA) in which the antigen membrane protein on cells was detected without inactivating endogenous enzymes, a pretreatment required for cell ELISA using conventional amplification enzymes. Here, we also developed a fluorescent substrate for α-SQase whose active residue is located at the end of the narrow, deep pocket of the substrate recognition site. The designed methylumbelliferyl substrate with a hydroxyl benzyl alcohol linker showed a similar reactivity to the p-nitrophenol substrate, a good substrate for α-SQase.

DOI： 10.18494/SAM4816

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記述言語：英語   出版者・発行元：Biochemical and Biophysical Research Communications  

Glucosinolates (GSLs), a class of secondary metabolites found in Brassicaceae plants, play important roles in plant defense and contribute distinct flavors and aromas when used as food ingredients. Following tissue damage, GSLs undergo enzymatic hydrolysis to release bioactive volatile compounds. Understanding GSL biosynthesis and enzyme involvement is crucial for improving crop quality and advancing agriculture. Plant sulfotransferases (SOTs) play a key role in the final step of GSL biosynthesis by transferring sulfate groups to the precursor molecules. In the present study, we investigated the enzymatic reaction mechanism and broad substrate specificity of Arabidopsis thaliana sulfotransferase AtSOT16, which is involved in GSL biosynthesis, using crystal structure analysis. Our analysis revealed the specific catalytic residues involved in the sulfate transfer reaction and supported the hypothesis of a concerted acid-base catalytic mechanism. Furthermore, the docking models showed a strong correlation between the substrates with high predicted binding affinities and those experimentally reported to exhibit high activity. These findings provide valuable insights into the enzymatic reaction mechanisms and substrate specificity of GSL biosynthesis. The information obtained in this study may contribute to the development of novel strategies for manipulating GSL synthesis pathways in Brassica plants and has potential agricultural applications.

DOI： 10.1016/j.bbrc.2023.08.020

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PubMed

担当区分：責任著者   記述言語：英語   出版者・発行元：Biochemical and Biophysical Research Communications  

The 3′-phosphoadenosine-5′-phosphosulfate (PAPS) molecule is essential during enzyme-catalyzed sulfation reactions as a sulfate donor and is an intermediate in the reduction of sulfate to sulfite in the sulfur assimilation pathway. PAPS is produced through a two-step reaction involving ATP sulfurylase and adenosine 5′-phosphosulfate (APS) kinase enzymes/domains. However, archaeal APS kinases have not yet been characterized and their mechanism of action remains unclear. Here, we first structurally characterized APS kinase from the hyperthermophilic archaeon Archaeoglobus fulgidus, (AfAPSK). We demonstrated the PAPS production activity of AfAPSK at the optimal growth temperature (83 °C). Furthermore, we determined the two crystal structures of AfAPSK: ADP complex and ATP analog adenylyl-imidodiphosphate (AMP-PNP)/Mg2+/APS complex. Structural and complementary mutational analyses revealed the catalytic and substrate recognition mechanisms of AfAPSK. This study also hints at the molecular basis behind the thermal stability of AfAPSK.

DOI： 10.1016/j.bbrc.2022.12.081

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PubMed

記述言語：英語   出版者・発行元：Cells  

RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, such as siRNA- and RNA-targeting CRISPR/Cas. Here, we demonstrate a versatile RNA manipulation technology using pentatricopeptide-repeat (PPR)-motif-containing proteins. First, we developed a rapid construction and evaluation method for PPR-based designer sequence-specific RNA-binding proteins. This system has enabled the steady construction of dozens of functional designer PPR proteins targeting long 18 nt RNA, which targets a single specific RNA in the mammalian transcriptome. Furthermore, the cellular functionality of the designer PPR proteins was first demonstrated by the control of alternative splicing of either a reporter gene or an endogenous CHK1 mRNA. Our results present a versatile protein-based RNA manipulation technology using PPR proteins that facilitates the understanding of unknown RNA functions and the creation of gene circuits and has potential for use in future therapeutics.

DOI： 10.3390/cells11223529

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Biochemistry  

The human estrogen-related receptor γ(ERRγ) is an orphan nuclear receptor. The ERRγbehaves as a constitutive activator of transcription and plays a key role in controlling mitochondrial energy production and energy metabolism. Bisphenol A (BPA) is used mainly in producing polycarbonate plastics and epoxy resins, but it is known as an endocrine disruptor and strongly binds to ERRγ. We determined the crystal structure of ERRγin complex with BPA. Our structure revealed the molecular mechanism of BPA recognition by ERRγ, in which BPA is well anchored to its ligand-binding pocket. Our structure is the first report of the complex between a nuclear receptor and endocrine disruptor BPA. This structural analysis had a profound impact on subsequent studies of endocrine disruptors.

DOI： 10.1093/jb/mvab145

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1261/rna.075416.120

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/2211-5463.12837

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.RA120.012491

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbagen.2017.08.005

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.7554/eLife.16381

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.febslet.2009.08.016

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbrc.2009.03.146

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbrc.2008.12.013

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.febslet.2008.10.035

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbrc.2008.06.050

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/jb/mvm158

開催年月日： 2024年5月

記述言語：日本語  

開催地：高知市   国名：日本国  

開催年月日： 2024年5月

記述言語：日本語  

開催地：福岡市   国名：日本国  

開催年月日： 2024年5月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：福岡市   国名：日本国  

放線菌Streptomyces sp.は、結核菌に対して強い抗菌作用を有する抗生物質カプラザマイシンを合成する。合成されたカプラザマイシンには硫酸化修飾された分子が一定数存在し、構造多様性を生み出す要因になっている。この修飾は、2種類の硫酸転移酵素(Cpz4とCpz8)により行われる。これら2つの硫酸転移酵素による2段階の硫酸化メカニズムは、まずCpz8が一般的な硫酸基ドナー分子PAPS依存的に硫酸基ドナー分子スルフィシジンを合成し、次にCpz4がそのスルフィシジンを硫酸基ドナー基質としてカプラザマイシンを硫酸化するものである。つまり、スルフィシジンは細胞内で硫酸基のシャトル分子として働いている。これらの分子メカニズムの詳細は、酵素の立体構造情報がないため、不明な点が多い。本研究は、カプラザマイシン硫酸化修飾に不可欠なCpz8、Cpz4の酵素反応メカニズムの詳細を明らかにするために、立体構造解析を行った。 Cpz8およびCpz4は、大腸菌組み換えタンパク質として発現し、精製した。立体構造決定は、X線結晶構造解析で行った。基質ドッキングモデル作成にはMOEを用いた。 Cpz8-PAP-スルフィシジン前駆体複合体について、分解能1.59Åで立体構造を決定した。スルフィシジン前駆体の六員環部分はAsp67, Trp108, Tyr120と水素結合しており、脂肪鎖は多数の疎水性残基によるトンネル状のポケットに囲まれていた。さらに、Arg10が一般酸触媒、Asp67が一般塩基触媒として逐次反応様式で硫酸転移反応が行われると考えられた。 Cpz4については、His308が硫酸化された反応中間体として、分解能2.11Åで立体構造を決定した。活性部位近傍には、小さな窪みを伴った広いポケットが存在し、基質ドッキングモデルで検討した結果、大きさの異なる2種類の基質(スルフィシジンとカプラザマイシン)をそれぞれ認識できると考えられた。さらに、His308が一時的に硫酸化される残基、His180とHis253は遷移状態を安定化する残基として働き、ピンポン反応様式で硫酸転移反応が行われると考えられた。 以上、2段階硫酸化の詳細な分子メカニズムを初めて明らかにすることができた。これらの情報は、抗生物質カプラザマイシンの構造多様性を生み出すことにつながる可能性がある。一方、本研究は細胞内の真の硫酸基シャトル分子の認識様式を明らかにした初めての報告であり、広範囲のバクテリアで行われている硫酸化反応の理解に大きく貢献するものだと考えられる。

開催年月日： 2024年5月

記述言語：日本語  

開催地：福岡市   国名：日本国  

開催年月日： 2024年5月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：福岡市   国名：日本国  

開催年月日： 2024年5月

記述言語：日本語  

国名：日本国  

開催年月日： 2024年5月

記述言語：英語   会議種別：シンポジウム・ワークショップ パネル（公募）  

国名：日本国  

開催年月日： 2024年5月

記述言語：英語   会議種別：シンポジウム・ワークショップ パネル（公募）  

国名：日本国  

開催年月日： 2023年9月 - 2022年9月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：長崎大学   国名：日本国  

開催年月日： 2023年8月

記述言語：英語  

国名：オーストラリア連邦  

開催年月日： 2023年7月

記述言語：日本語  

国名：日本国  

開催年月日： 2023年7月

記述言語：日本語  

開催地：北九州国際会議場   国名：日本国  

開催年月日： 2023年7月

記述言語：日本語  

開催地：北九州国際会議場   国名：日本国  

開催年月日： 2023年6月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：オンライン   国名：日本国  

開催年月日： 2023年6月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：オンライン   国名：日本国  

開催年月日： 2022年11月

記述言語：英語   会議種別：口頭発表（一般）  

国名：日本国  

開催年月日： 2022年11月

記述言語：英語   会議種別：口頭発表（一般）  

国名：日本国  

開催年月日： 2022年10月

記述言語：英語   会議種別：口頭発表（一般）  

国名：大韓民国  

開催年月日： 2022年10月

記述言語：英語   会議種別：口頭発表（一般）  

国名：大韓民国  

開催年月日： 2022年10月

記述言語：英語   会議種別：口頭発表（一般）  

国名：大韓民国  

開催年月日： 2022年9月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：長崎大学   国名：日本国  

開催年月日： 2022年9月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：長崎大学   国名：日本国  

開催年月日： 2022年9月

記述言語：日本語   会議種別：口頭発表（一般）  

国名：日本国  

開催年月日： 2022年8月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：長崎市   国名：日本国  

開催年月日： 2022年7月

記述言語：日本語  

開催地：北九州国際会議場   国名：日本国  

開催年月日： 2022年7月

記述言語：日本語  

開催地：北九州国際会議場   国名：日本国  

開催年月日： 2022年1月

記述言語：日本語   会議種別：口頭発表（一般）  

国名：日本国  

開催年月日： 2021年9月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

国名：日本国  

開催年月日： 2021年7月

記述言語：日本語  

開催地：オンライン   国名：日本国  

開催年月日： 2021年7月

記述言語：日本語  

国名：日本国  

開催年月日： 2021年7月

記述言語：日本語  

国名：日本国  

開催年月日： 2021年6月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

国名：日本国  

開催年月日： 2020年11月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：宮崎大学   国名：日本国  

開催年月日： 2020年5月

記述言語：日本語  

国名：日本国  

記述言語：日本語  

記述言語：日本語  

記述言語：日本語  

記述言語：日本語  

記述言語：日本語