記述言語：英語   掲載種別：研究論文（学術雑誌）  

Para-hydroxybenzoate hydroxylase (PHBH) is a group A flavoprotein monooxygenase that hydroxylates p-hydroxybenzoate to protocatechuate (PCA). Despite intensive studies of Pseudomonas aeruginosa p-hydroxybenzoate hydroxylase (PaPobA), the catalytic reactions of extremely diverse putative PHBH isozymes remain unresolved. We analyzed the phylogenetic relationships of known and predicted PHBHs and identified eight divergent clades. Clade F contains a protein that lacks the critical amino acid residues required for PaPobA to generate PHBH activity. Among proteins in this clade, Xylophilus ampelinus PobA (XaPobA) preferred PCA as a substrate and is the first known natural PCA 5-hydroxylase (PCAH). Crystal structures and kinetic properties revealed similar mechanisms of substrate carboxy group recognition between XaPobA and PaPobA. The unique Ile75, Met72, Val199, Trp201, and Phe385 residues of XaPobA form the bottom of a hydrophobic cavity with a shape that complements the 3-and 4-hydroxy groups of PCA and its binding site configuration. An interaction between the δ-sulfur atom of Met210 and the aromatic ring of PCA is likely to stabilize XaPobA-PCA complexes. The 4-hydroxy group of PCA forms a hydrogen bond with the main chain carbonyl of Thr294. These modes of binding constitute a novel substrate recognition mechanism that PaPobA lacks. This mechanism characterizes XaPobA and sheds light on the diversity of catalytic mechanisms of PobA-type PHBHs and group A flavoprotein monooxygenases.

DOI： 10.1016/j.jbc.2023.105508

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掲載種別：研究論文（学術雑誌）   出版者・発行元：The Japanese Society of Applied Glycoscience  

DOI： 10.5458/bag.13.4_194

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The cell walls of pathogenic and acidophilic bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae, contain lipoarabinomannan and arabinogalactan. These components are composed of D-arabinose, the enantiomer of the typical L-arabinose found in plants. The unique glycan structures of mycobacteria contribute to their ability to evade mammalian immune responses. In this study, we identified four enzymes (two GH183 endo-D-arabinanases, GH172 exo-α-D-arabinofuranosidase, and GH116 exo-β-D-arabinofuranosidase) from Microbacterium arabinogalactanolyticum. These enzymes completely degraded the complex D-arabinan core structure of lipoarabinomannan and arabinogalactan in a concerted manner. Furthermore, through biochemical characterization using synthetic substrates and X-ray crystallography, we elucidated the mechanisms of substrate recognition and anomer-retaining hydrolysis for the α- and β-D-arabinofuranosidic bonds in both endo- and exo-mode reactions. The discovery of these D-arabinan-degrading enzymes, along with the understanding of their structural basis for substrate specificity, provides valuable resources for investigating the intricate glycan architecture of mycobacterial cell wall polysaccharides and their contribution to pathogenicity.

DOI： 10.1038/s41467-023-41431-2

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mucinolytic bacteria modulate host-microbiota symbiosis and dysbiosis through their ability to degrade mucin O-glycans. However, how and to what extent bacterial enzymes are involved in the breakdown process remains poorly understood. Here we focus on a glycoside hydrolase family 20 sulfoglycosidase (BbhII) from Bifidobacterium bifidum, which releases N-acetylglucosamine-6-sulfate from sulfated mucins. Glycomic analysis showed that, in addition to sulfatases, sulfoglycosidases are involved in mucin O-glycan breakdown in vivo and that the released N-acetylglucosamine-6-sulfate potentially affects gut microbial metabolism, both of which were also supported by a metagenomic data mining analysis. Enzymatic and structural analysis of BbhII reveals the architecture underlying its specificity and the presence of a GlcNAc-6S-specific carbohydrate-binding module (CBM) 32 with a distinct sugar recognition mode that B. bifidum takes advantage of to degrade mucin O-glycans. Comparative analysis of the genomes of prominent mucinolytic bacteria also highlights a CBM-dependent O-glycan breakdown strategy used by B. bifidum.

DOI： 10.1038/s41589-023-01272-y

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

β-l-Arabinofuranosidase HypBA1 from Bifidobacterium longum belongs to the glycoside hydrolase family 127. At the active site of HypBA1, a cysteine residue (Cys417) coordinates with a Zn2+ atom and functions as the catalytic nucleophile for the anomer-retaining hydrolytic reaction. In this study, the role of Zn2+ ion and cysteine in catalysis as well as the substrate-bound structure were studied based on biochemical and crystallographic approaches. The enzymatic activity of HypBA1 decreased after dialysis in the presence of EDTA and guanidine hydrochloride and was then recovered by the addition of Zn2+. The Michaelis complex structure was determined using a crystal of a mutant at the acid/base catalyst residue (E322Q) soaked in a solution containing the substrate p-nitrophenyl-β-l-arabinofuranoside. To investigate the covalent thioglycosyl enzyme intermediate structure, synthetic inhibitors of l-arabinofuranosyl haloacetamide derivatives with different anomer configurations were used to target the nucleophilic cysteine. In the crystal structure of HypBA1, β-configured l-arabinofuranosylamide formed a covalent link with Cys417, whereas α-configured l-arabinofuranosylamide was linked to a noncatalytic residue Cys415. Mass spectrometric analysis indicated that Cys415 was also reactive with the probe molecule. With the β-configured inhibitor, the arabinofuranoside moiety was correctly positioned at the subsite and the active site integrity was retained to successfully mimic the covalent intermediate state.

DOI： 10.1093/glycob/cwab103

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press ({OUP})  

<jats:title>Abstract</jats:title>
<jats:p>Glycoside hydrolase family 136 (GH136) was established after the discovery and structural analysis of lacto-N-biosidase (LNBase) from the infant gut bacterium Bifidobacterium longum subsp. longum JCM1217 (BlLnbX). Homologous genes of BlLnbX are widely distributed in the genomes of human gut bacteria and monkey Bifidobacterium spp., although only two crystal structures were reported in the GH136 family. Cell suspensions of Bifidobacterium saguini, Tyzzerella nexilis, and Ruminococcus lactaris exhibited the LNBase activity. Recombinant LNBases of these three species were functionally expressed with their specific chaperones in Escherichia coli, and their kinetic parameters against p-nitrophenol substrates were determined. The crystal structures of the LNBases from B. saguini and T. nexilis in complex with lacto-N-biose I were determined at 2.5 Å and 1.9 Å resolutions, respectively. These structures conserve a β-helix fold characteristic of GH136 and the catalytic residues, but they lack the metal ions that were present in BlLnbX.</jats:p>

DOI： 10.1093/bbb/zbac015

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.jbc.2021.101324

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3 (Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived β-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This β-l-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.

DOI： 10.1002/anie.202013920

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

DOI： 10.1038/s41598-020-60002-9

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41467-020-17075-x

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.0231513

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.yjsbx.2020.100030

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Biochemistry {\&} Molecular Biology ({ASBMB})  

DOI： 10.1074/jbc.RA119.010842

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acs.biochem.9b00443

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1038/s41598-018-30508-4

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その他リンク： http://www.nature.com/articles/s41598-018-30508-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.RA118.004963

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-018-32080-3

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.chembiol.2017.03.012

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その他リンク： http://orcid.org/0000-0002-0083-1838

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.2323/jgam.2016.06.002

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jbiosc.2014.11.001

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/prot.24649

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Three thermophilic methanogens (Methanothermobacter thermautotrophicus, Methanosaeta thermophila, and Methanosarcina thermophila) were investigated for their ability to reduce poorly crystalline Fe(III) oxides (ferrihydrite) and the inhibitory effects of ferrihydrite on their methanogenesis. This study demonstrated that Fe(II) generation from ferrihydrite occurs in the cultures of the three thermophilic methanogens only when H2 was supplied as the source of reducing equivalents, even in the cultures of Mst. thermophila that do not grow on and produce CH4 from H2/CO2. While supplementation of ferrihydrite resulted in complete inhibition or suppression of methanogenesis by the thermophilic methanogens, ferrihydrite reduction by the methanogens at least partially alleviates the inhibitory effects. Microscopic and crystallographic analyses on the ferrihydrite-reducing Msr. thermophila cultures exhibited generation of magnetite on its cell surfaces through partial reduction of ferrihydrite. These findings suggest that at least certain thermophilic methanogens have the ability to extracellularly transfer electrons to insoluble Fe(III) compounds, affecting their methanogenic activities, which would in turn have significant impacts on materials and energy cycles in thermophilic anoxic environments.

DOI： 10.1111/1574-6941.12365

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The addition of ferrihydrite to methanogenic microbial communities obtained from a thermophilic anaerobic digester suppressed methanogenesis in a dose-dependent manner. The amount of reducing equivalents consumed by the reduction of iron was significantly smaller than that expected from the decrease in the production of CH4, which suggested that competition between iron-reducing microorganisms and methanogens was not the most significant cause for the suppression of methanogenesis. Microbial community analyses revealed that the presence of ferrihydrite markedly affected the bacterial composition, but not the archaeal composition. These results indicate that the presence of ferrihydrite directly and indirectly suppresses thermophilic methanogenesis.

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.15625/0866-7160/v34n3se.1785

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J-GLOBAL

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記述言語：日本語   出版者・発行元：日本応用糖質科学会  

CiNii Research

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その他リンク： http://search.jamas.or.jp/link/ui/2017251609

J-GLOBAL

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記述言語：日本語   出版者・発行元：日本生物工学会  

CiNii Research

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記述言語：日本語   出版者・発行元：日本生物工学会  

CiNii Research

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