Publications by authors named "Shinya Fushinobu"

162 Publications

Substrate complex structure, active site labeling and catalytic role of the zinc ion in cysteine glycosidase.

Glycobiology 2021 Oct 7. Epub 2021 Oct 7.

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

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.
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http://dx.doi.org/10.1093/glycob/cwab103DOI Listing
October 2021

Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family.

J Biol Chem 2021 11 22;297(5):101324. Epub 2021 Oct 22.

Department of Biotechnology, The University of Tokyo, Tokyo, Japan; Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan. Electronic address:

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of β-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2':2,1'-β-D-Frup (diheterolevulosan II) and β-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2':2,1'-β-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with β-D-Fruf and β-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).
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http://dx.doi.org/10.1016/j.jbc.2021.101324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8605356PMC
November 2021

Structural and functional analysis of gum arabic l-rhamnose-α-1,4-d-glucuronate lyase establishes a novel polysaccharide lyase family.

J Biol Chem 2021 09 23;297(3):101001. Epub 2021 Jul 23.

Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan. Electronic address:

Gum arabic (GA) is widely used as an emulsion stabilizer and coating in several industrial applications, such as foods and pharmaceuticals. GA contains a complex carbohydrate moiety, and the nonreducing ends of the side chains are often capped with l-rhamnose; thus, enzymes that can remove these caps are promising tools for the structural analysis of the carbohydrates comprising GA. In this study, GA-specific l-rhamnose-α-1,4-d-glucuronate lyase from the fungus Fusarium oxysporum 12S (FoRham1) was cloned and characterized. FoRham1 showed the highest amino acid sequence similarity with enzymes belonging to the glycoside hydrolase family 145; however, the catalytic residue on the posterior pocket of the β-propeller fold protein was not conserved. The catalytic residues of FoRham1 were instead conserved with ulvan lyases belonging to polysaccharide lyase family 24. Kinetic analysis showed that FoRham1 has the highest catalytic efficiency for the substrate α-l-rhamnose-(1→4)-d-glucuronic acid. The crystal structures of ligand-free and α-l-rhamnose-(1→4)-d-glucuronic acid -bound FoRham1 were determined, and the active site was identified on the anterior side of the β-propeller. The three-dimensional structure of the active site and mutagenesis analysis revealed the detailed catalytic mechanism of FoRham1. Our findings offer a new enzymatic tool for the further analysis of the GA carbohydrate structure and for elucidating its physiological functions in plants. Based on these results, we renamed glycoside hydrolase family 145 as a new polysaccharide lyase family 42, in which FoRham1 is included.
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http://dx.doi.org/10.1016/j.jbc.2021.101001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8377490PMC
September 2021

Structure and evolution of the bifidobacterial carbohydrate metabolism proteins and enzymes.

Biochem Soc Trans 2021 04;49(2):563-578

Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800 Lyngby, Denmark.

Bifidobacteria have attracted significant attention because they provide health-promoting effects in the human gut. In this review, we present a current overview of the three-dimensional structures of bifidobacterial proteins involved in carbohydrate uptake, degradation, and metabolism. As predominant early colonizers of the infant's gut, distinct bifidobacterial species are equipped with a panel of transporters and enzymes specific for human milk oligosaccharides (HMOs). Interestingly, Bifidobacterium bifidum and Bifidobacterium longum possess lacto-N-biosidases with unrelated structural folds to release the disaccharide lacto-N-biose from HMOs, suggesting the convergent evolution of this activity from different ancestral proteins. The crystal structures of enzymes that confer the degradation of glycans from the mucin glycoprotein layer provide a structural basis for the utilization of this sustainable nutrient in the gastrointestinal tract. The utilization of several plant dietary oligosaccharides has been studied in detail, and the prime importance of oligosaccharide-specific ATP-binding cassette (ABC) transporters in glycan utilisations by bifidobacteria has been revealed. The structural elements underpinning the high selectivity and roles of ABC transporter binding proteins in establishing competitive growth on preferred oligosaccharides are discussed. Distinct ABC transporters are conserved across several bifidobacterial species, e.g. those targeting arabinoxylooligosaccharide and α-1,6-galactosides/glucosides. Less prevalent transporters, e.g. targeting β-mannooligosaccharides, may contribute to the metabolic specialisation within Bifidobacterium. Some bifidobacterial species have established symbiotic relationships with humans. Structural studies of carbohydrate-utilizing systems in Bifidobacterium have revealed the interesting history of molecular coevolution with the host, as highlighted by the early selection of bifidobacteria by mucin and breast milk glycans.
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http://dx.doi.org/10.1042/BST20200163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106489PMC
April 2021

Biochemical and structural characterization of a novel 4-O-α-l-rhamnosyl-β-d-glucuronidase from Fusarium oxysporum.

FEBS J 2021 08 11;288(16):4918-4938. Epub 2021 Mar 11.

Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan.

In this study, we have isolated the novel enzyme 4-O-α-l-rhamnosyl-β-d-glucuronidase (FoBGlcA), which releases α-l-rhamnosyl (1→4) glucuronic acid from gum arabic (GA), from Fusarium oxysporum 12S culture supernatant, and for the first time report an enzyme with such catalytic activity. The gene encoding FoBGlcA was cloned and expressed in Pichia pastoris. When GA was subjected to the recombinant enzyme, > 95% of the l-rhamnose (Rha) and d-glucuronic acid in the substrate were released, which indicates that almost all Rha binds to the glucuronic acid at the end of the GA side chains. The crystal structure of FoBGlcA was determined using a single-wavelength anomalous dispersion at 1.51 Å resolution. FoBGlcA consisted of an N-terminal (β/α) -barrel domain and a C-terminal antiparallel β-sheet domain. This configuration is characteristic of glycoside hydrolase (GH) family 79 proteins. A structural similarity search showed that FoBGlcA mostly resembled GH79 β-d-glucuronidase (AcGlcA79A) of Acidobacterium capsulatum; however, the root-mean-square deviation value was 3.2 Å, indicating that FoBGlcA has a high structural divergence. FoBGlcA had a low sequence identity with AcGlcA79A (19%) and differed from other GH79 β-glucuronidases. The structures of FoBGlcA and AcGlcA79A also differed in terms of the loop structure location near subsite -2 of their catalytic sites, which may account for the unique substrate specificity of FoBGlcA. The amino acid residues involved in the catalytic activity of this enzyme were determined by evaluating the activity levels of various mutant enzymes based on the crystal structure analysis of the FoBGlcA reaction product complex. DATABASE: Atomic coordinates and structure factors (codes 7DFQ and 7DFS) have been deposited in the Protein Data Bank (http://wwpdb.org/).
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http://dx.doi.org/10.1111/febs.15795DOI Listing
August 2021

Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent β-l-Arabinofuranosidase Inhibitor.

Angew Chem Int Ed Engl 2021 03 2;60(11):5754-5758. Epub 2021 Feb 2.

York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York, YO10 5DD, UK.

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys) (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.
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http://dx.doi.org/10.1002/anie.202013920DOI Listing
March 2021

Molecular analysis of cyclic α-maltosyl-(1→6)-maltose binding protein in the bacterial metabolic pathway.

PLoS One 2020 19;15(11):e0241912. Epub 2020 Nov 19.

Department of Biotechnology, The University of Tokyo, Tokyo, Japan.

Cyclic α-maltosyl-(1→6)-maltose (CMM) is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. Here, we report functional and structural analyses on CMM-binding protein (CMMBP), which is a substrate-binding protein (SBP) of an ABC importer system of the bacteria Arthrobacter globiformis. Isothermal titration calorimetry analysis revealed that CMMBP specifically bound to CMM with a Kd value of 9.6 nM. The crystal structure of CMMBP was determined at a resolution of 1.47 Å, and a panose molecule was bound in a cleft between two domains. To delineate its structural features, the crystal structure of CMMBP was compared with other SBPs specific for carbohydrates, such as cyclic α-nigerosyl-(1→6)-nigerose and cyclodextrins. These results indicate that A. globiformis has a unique metabolic pathway specialized for CMM.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241912PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676653PMC
January 2021

Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction.

Sci Rep 2020 11 18;10(1):20066. Epub 2020 Nov 18.

Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, 3-5-1 Johoku, Naka-ku, Hamamatsu, 432-8561, Japan.

Levoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes β-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-D-glucose by LgdB2; next, LgdC reduces 3-keto-D-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.
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http://dx.doi.org/10.1038/s41598-020-77133-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676230PMC
November 2020

Molecular evolution and functional divergence of UDP-hexose 4-epimerases.

Authors:
Shinya Fushinobu

Curr Opin Chem Biol 2021 04 7;61:53-62. Epub 2020 Nov 7.

Department of Biotechnology, The University of Tokyo, Tokyo, 113-8657, Japan; Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, 113-8657, Japan. Electronic address:

UDP-glucose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) and/or the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) in sugar metabolism. GalEs belong to the short-chain dehydrogenase/reductase superfamily, use a conserved 'transient keto intermediate' mechanism and have variable substrate specificity. GalEs have been classified into three groups based on substrate specificity: group 1 prefers UDP-Glc/Gal, group 3 prefers UDP-GlcNAc/GalNAc, and group 2 has comparable activities for both types of the substrates. The phylogenetic relationship and structural basis for the specificities of GalEs revealed possible molecular evolution of UDP-hexose 4-epimerases in various organisms. Based on the recent advances in studies on GalEs and related enzymes, an updated view of their evolutional diversification is presented.
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http://dx.doi.org/10.1016/j.cbpa.2020.09.007DOI Listing
April 2021

Crystallographic and cryogenic electron microscopic structures and enzymatic characterization of sulfur oxygenase reductase from .

J Struct Biol X 2020 3;4:100030. Epub 2020 Jul 3.

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

Sulfur oxygenase reductases (SORs) are present in thermophilic and mesophilic archaea and bacteria, and catalyze oxygen-dependent oxygenation and disproportionation of elemental sulfur. SOR has a hollow, spherical homo-24-mer structure and reactions take place at active sites inside the chamber. The crystal structures of SORs from species have been reported. However, the states of the active site components (mononuclear iron and cysteines) and the entry and exit paths of the substrate and products are still in dispute. Here, we report the biochemical and structural characterizations of SORs from the thermoacidophilic archaeon (StSOR) and present high-resolution structures determined by X-ray crystallography and cryogenic electron microscopy (cryo-EM). The crystal structure of StSOR was determined at 1.73 Å resolution. At the catalytic center, iron is ligated to His86, His90, Glu114, and two water molecules. Three conserved cysteines in the cavity are located 9.5-13 Å from the iron and were observed as free thiol forms. A mutational analysis indicated that the iron and one of the cysteines (Cys31) were essential for both activities. The cryo-EM structure was determined at 2.24 Å resolution using an instrument operating at 200 kV. The two structures determined by different methodologies showed similar main chain traces, but the maps exhibited different features at catalytically important components. A possible role of StSOR in the sulfur metabolism of (an obligate aerobe) is discussed based on this study. Given the high resolution achieved in this study, StSOR was shown to be a good benchmark sample for cryo-EM.
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http://dx.doi.org/10.1016/j.yjsbx.2020.100030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398979PMC
July 2020

Butyrate producing colonic Clostridiales metabolise human milk oligosaccharides and cross feed on mucin via conserved pathways.

Nat Commun 2020 07 3;11(1):3285. Epub 2020 Jul 3.

Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800, Lyngby, Denmark.

The early life human gut microbiota exerts life-long health effects on the host, but the mechanisms underpinning its assembly remain elusive. Particularly, the early colonization of Clostridiales from the Roseburia-Eubacterium group, associated with protection from colorectal cancer, immune- and metabolic disorders is enigmatic. Here, we describe catabolic pathways that support the growth of Roseburia and Eubacterium members on distinct human milk oligosaccharides (HMOs). The HMO pathways, which include enzymes with a previously unknown structural fold and specificity, were upregulated together with additional glycan-utilization loci during growth on selected HMOs and in co-cultures with Akkermansia muciniphila on mucin, suggesting an additional role in enabling cross-feeding and access to mucin O-glycans. Analyses of 4599 Roseburia genomes underscored the preponderance and diversity of the HMO utilization loci within the genus. The catabolism of HMOs by butyrate-producing Clostridiales may contribute to the competitiveness of this group during the weaning-triggered maturation of the microbiota.
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http://dx.doi.org/10.1038/s41467-020-17075-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335108PMC
July 2020

Crystal structure of β-L-arabinobiosidase belonging to glycoside hydrolase family 121.

PLoS One 2020 1;15(6):e0231513. Epub 2020 Jun 1.

Department of Biotechnology, The University of Tokyo, Tokyo, Japan.

Enzymes acting on α-L-arabinofuranosides have been extensively studied; however, the structures and functions of β-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of β-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular β-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing β-1,2-linked arabinofuranose disaccharide (β-Ara2) for the specific sugar importer. Here, we present the crystal structure of the catalytic region of HypBA2 as the first three-dimensional structure of GH121 at 1.85 Å resolution. The HypBA2 structure consists of a central catalytic (α/α)6 barrel domain and two flanking (N- and C-terminal) β-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the β-Ara2 disaccharide. Three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all members of GH121; site-directed mutagenesis analysis showed that they are essential for catalytic activity. The active site of HypBA2 was compared with those of structural homologs in other GH families: GH63 α-glycosidase, GH94 chitobiose phosphorylase, GH142 β-L-arabinofuranosidase, GH78 α-L-rhamnosidase, and GH37 α,α-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. β-L-Arabinobiosidase genes in GH121 are mainly found in the genomes of bifidobacteria and Xanthomonas species, suggesting that the cleavage and specific import system for the β-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231513PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263609PMC
August 2020

Structural basis for selectivity in a highly reducing type II polyketide synthase.

Nat Chem Biol 2020 07 4;16(7):776-782. Epub 2020 May 4.

Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

In type II polyketide synthases (PKSs), the ketosynthase-chain length factor (KS-CLF) complex catalyzes polyketide chain elongation with the acyl carrier protein (ACP). Highly reducing type II PKSs, represented by IgaPKS, produce polyene structures instead of the well-known aromatic skeletons. Here, we report the crystal structures of the Iga11-Iga12 (KS-CLF) heterodimer and the covalently cross-linked Iga10=Iga11-Iga12 (ACP=KS-CLF) tripartite complex. The latter structure revealed the molecular basis of the interaction between Iga10 and Iga11-Iga12, which differs from that between the ACP and KS of Escherichia coli fatty acid synthase. Furthermore, the reaction pocket structure and site-directed mutagenesis revealed that the negative charge of Asp 113 of Iga11 prevents further condensation using a β-ketoacyl product as a substrate, which distinguishes IgaPKS from typical type II PKSs. This work will facilitate the future rational design of PKSs.
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http://dx.doi.org/10.1038/s41589-020-0530-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556716PMC
July 2020

Structural analysis of β-L-arabinobiose-binding protein in the metabolic pathway of hydroxyproline-rich glycoproteins in Bifidobacterium longum.

FEBS J 2020 12 17;287(23):5114-5129. Epub 2020 Apr 17.

Department of Biotechnology, The University of Tokyo, Japan.

Bifidobacterium longum is a symbiotic human gut bacterium that has a degradation system for β-arabinooligosaccharides, which are present in the hydroxyproline-rich glycoproteins of edible plants. Whereas microbial degradation systems for α-linked arabinofuranosyl carbohydrates have been extensively studied, little is understood about the degradation systems targeting β-linked arabinofuranosyl carbohydrates. We functionally and structurally analyzed a substrate-binding protein (SBP) of a putative ABC transporter (BLLJ_0208) in the β-arabinooligosaccharide degradation system. Thermal shift assays and isothermal titration calorimetry revealed that the SBP specifically bound Araf-β1,2-Araf (β-Ara ) with a K of 0.150 μm, but did not bind L-arabinose or methyl-β-Ara . Therefore, the SBP was termed β-arabinobiose-binding protein (BABP). Crystal structures of BABP complexed with β-Ara were determined at resolutions of up to 1.78 Å. The findings showed that β-Ara was bound to BABP within a short tunnel between two lobes as an α-anomeric form at its reducing end. BABP forms extensive interactions with β-Ara , and its binding mode was unique among SBPs. A molecular dynamics simulation revealed that the closed conformation of substrate-bound BABP is stable, whereas substrate-free form can adopt a fully open and two distinct semi-open states. The importer system specific for β-Ara may contribute to microbial survival in biological niches with limited amounts of digestible carbohydrates. DATABASE: Atomic coordinates and structure factors (codes 6LCE and 6LCF) have been deposited in the Protein Data Bank (http://wwpdb.org/).
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http://dx.doi.org/10.1111/febs.15315DOI Listing
December 2020

Streptococcus pneumoniae hijacks host autophagy by deploying CbpC as a decoy for Atg14 depletion.

EMBO Rep 2020 05 2;21(5):e49232. Epub 2020 Apr 2.

Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan.

Pneumococcal cell surface-exposed choline-binding proteins (CBPs) play pivotal roles in multiple infectious processes with pneumococci. Intracellular pneumococci can be recognized at multiple steps during bactericidal autophagy. However, whether CBPs are involved in pneumococci-induced autophagic processes remains unknown. In this study, we demonstrate that CbpC from S. pneumoniae strain TIGR4 activates autophagy through an interaction with Atg14. However, S. pneumoniae also interferes with autophagy by deploying CbpC as a decoy to cause autophagic degradation of Atg14 through an interaction with p62/SQSTM1. Thus, S. pneumoniae suppresses the autophagic degradation of intracellular pneumococci and survives within cells. Domain analysis reveals that the coiled-coil domain of Atg14 and residue Y83 of the dp3 domain in the N-terminal region of CbpC are crucial for both the CbpC-Atg14 interaction and the subsequent autophagic degradation of Atg14. Although homology modeling indicates that CbpC orthologs have similar structures in the dp3 domain, autophagy induction through Atg14 binding is an intrinsic property of CbpC Our data provide novel insights into the evolutionary hijacking of host-defense systems by intracellular pneumococci.
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http://dx.doi.org/10.15252/embr.201949232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202210PMC
May 2020

Author Correction: Identification and characterization of a novel β-D-galactosidase that releases pyruvylated galactose.

Sci Rep 2020 Feb 13;10(1):2873. Epub 2020 Feb 13.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-60002-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016099PMC
February 2020

Regio- and stereoselective hydroxylation of testosterone by a novel cytochrome P450 154C2 from Streptomyces avermitilis.

Biochem Biophys Res Commun 2020 02 22;522(2):355-361. Epub 2019 Nov 22.

College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, Zhejiang, 310018, China. Electronic address:

Cytochrome P450 enzymes (P450 or CYP) are some of the most versatile biocatalysts, and offer advantages for oxidizing unreactive C-H bonds in mild conditions. In this study, we identified a novel cytochrome P450 154C2 from Streptomyces avermitilis and characterized its function in 2α-hydroxylation of testosterone with regio- and stereoselectivity. To investigate the efficiency of electron transfer, we conducted biotransformation using two different P450 redox partners-RhFRED (RhF reductase domain) from Rhodococcus sp. and Pdx (putidaredoxin)/Pdr (putidaredoxin reductase) from Pseudomonas putida and revealed that RhFRED was more effective than Pdx/Pdr, especially in vivo. The K and k values for testosterone were estimated to be 0.16 ± 0.05 mM and 0.13 ± 0.02 min, and k/K was 0.81 min mM. We also determined the crystal structure of the substrate-free form of CYP154C2 at 1.5 Å resolution. The structure has a closed conformation, and the substrate binding pocket is narrow, which can explain the strict substrate specificity of the enzyme.
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http://dx.doi.org/10.1016/j.bbrc.2019.11.091DOI Listing
February 2020

Understanding the Molecular Mechanism Underlying the High Catalytic Activity of -Hydroxybenzoate Hydroxylase Mutants for Producing Gallic Acid.

Biochemistry 2019 11 1;58(45):4543-4558. Epub 2019 Nov 1.

The Collaborative Research Institute for Innovative Microbiology , The University of Tokyo , 1-1-1 Yayoi , Bunkyo-ku, Tokyo 113-8657 , Japan.

-Hydroxybenzoate hydroxylase (PHBH) is a flavoprotein monooxygenase that catalyzes the hydroxylation of -hydroxybenzoate (-OHB) to 3,4-dihydroxybenzoate (3,4-DOHB). PHBH can bind to other benzoate derivatives in addition to -OHB; however, hydroxylation does not occur on 3,4-DOHB. Replacement of Tyr385 with Phe forms a mutant, which enables the production of 3,4,5-trihydroxybenzonate (gallic acid) from 3,4-DOHB, although the catalytic activity of the mutant is quite low. In this study, we report how the L199V/Y385F double mutant exhibits activity for producing gallic acid 4.3-fold higher than that of the Y385F single mutant. This improvement in catalytic activity is primarily due to the suppression of a shunt reaction that wastes reduced nicotinamide adenine dinucleotide phosphate by producing HO. To further elucidate the molecular mechanism underlying this higher catalytic activity, we performed molecular dynamics simulations and quantum mechanics/molecular mechanics calculations, in addition to determining the crystal structure of the Y385F·3,4-DOHB complex. The simulations showed that the Y385F mutation facilitates the deprotonation of the 4-hydroxy group of 3,4-DOHB, which is necessary for initiating hydroxylation. Moreover, the L199V mutation in addition to the Y385F mutation allows the OH moiety in the peroxide group of C-(4a)-flavin hydroperoxide to come into the proximity of the C5 atom of 3,4-DOHB. Overall, this study provides a consistent explanation for the change in the catalytic activity of PHBH caused by mutations, which will enable us to better design an enzyme with different activities.
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http://dx.doi.org/10.1021/acs.biochem.9b00443DOI Listing
November 2019

Structural basis for the specific cleavage of core-fucosylated -glycans by endo-β--acetylglucosaminidase from the fungus .

J Biol Chem 2019 11 23;294(45):17143-17154. Epub 2019 Sep 23.

Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

-Linked glycans play important roles in various cellular and immunological events. Endo-β--acetylglucosaminidase (ENGase) can release or transglycosylate -glycans and is a promising tool for the chemoenzymatic synthesis of glycoproteins with homogeneously modified glycans. The ability of ENGases to act on core-fucosylated glycans is a key factor determining their therapeutic utility because mammalian -glycans are frequently α-1,6-fucosylated. Although the biochemistries and structures of various ENGases have been studied extensively, the structural basis for the recognition of the core fucose and the asparagine-linked GlcNAc is unclear. Herein, we determined the crystal structures of a core fucose-specific ENGase from the caterpillar fungus (Endo-CoM), which belongs to glycoside hydrolase family 18. Structures complexed with fucose-containing ligands were determined at 1.75-2.35 Å resolutions. The fucose moiety linked to GlcNAc is extensively recognized by protein residues in a round-shaped pocket, whereas the asparagine moiety linked to the GlcNAc is exposed to the solvent. The -glycan-binding cleft of Endo-CoM is Y-shaped, and several lysine and arginine residues are present at its terminal regions. These structural features were consistent with the activity of Endo-CoM on fucose-containing glycans on rituximab (IgG) and its preference for a sialobiantennary substrate. Comparisons with other ENGases provided structural insights into their core fucose tolerance and specificity. In particular, Endo-F3, a known core fucose-specific ENGase, has a similar fucose-binding pocket, but the surrounding residues are not shared with Endo-CoM. Our study provides a foothold for protein engineering to develop enzymatic tools for the preparation of more effective therapeutic antibodies.
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http://dx.doi.org/10.1074/jbc.RA119.010842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851319PMC
November 2019

Structural basis for broad substrate specificity of UDP-glucose 4-epimerase in the human milk oligosaccharide catabolic pathway of Bifidobacterium longum.

Sci Rep 2019 07 31;9(1):11081. Epub 2019 Jul 31.

Department of Biotechnology, The University of Tokyo, Tokyo, 113-8657, Japan.

Infant gut-associated bifidobacteria has a metabolic pathway that specifically utilizes lacto-N-biose I (Gal-β1,3-GlcNAc) and galacto-N-biose (Gal-β1,3-GalNAc) from human milk and mucin glycans. UDP-glucose 4-epimerase (GalE) from Bifidobacterium longum (bGalE) catalyzes epimerization reactions of UDP-Gal into UDP-Glc and UDP-GalNAc into UDP-GlcNAc with the same level of activity that is required to send galacto-hexoses into glycolysis. Here, we determined the crystal structures of bGalE in three ternary complex forms: NAD/UDP, NAD/UDP-GlcNAc, and NAD/UDP-Glc. The broad specificity of bGalE was explained by structural features of the binding pocket for the N-acetyl or C2 hydroxy group of the substrate. Asn200 is located in a pocket of the C2 group, and its side chain adopts different conformations in the complex structures with UDP-Glc and UDP-GlcNAc. On the other side, Cys299 forms a large pocket for the C5 sugar ring atom. The flexible C2 pocket and the large C5 pocket of bGalE are suitable for accommodating both the hydroxy and N-acetyl groups of the substrate during sugar ring rotation in the catalytic cycle. The substrate specificity and active site structure of bGalE were distinct from those of Esherichia coli GalE but similar to those of human GalE.
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http://dx.doi.org/10.1038/s41598-019-47591-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668579PMC
July 2019

Identification, functional characterization, and crystal structure determination of bacterial levoglucosan dehydrogenase.

J Biol Chem 2018 11 17;293(45):17375-17386. Epub 2018 Sep 17.

From the Department of Biotechnology and

Levoglucosan is the 1,6-anhydrosugar of d-glucose formed by pyrolysis of glucans and is found in the environment and industrial waste. Two types of microbial levoglucosan metabolic pathways are known. Although the eukaryotic pathway involving levoglucosan kinase has been well-studied, the bacterial pathway involving levoglucosan dehydrogenase (LGDH) has not been well-investigated. Here, we identified and cloned the gene from the bacterium and characterized the recombinant protein. The enzyme exhibited high substrate specificity toward levoglucosan and NAD for the oxidative reaction and was confirmed to be LGDH. LGDH also showed weak activities (∼4%) toward l-sorbose and 1,5-anhydro-d-glucitol. The reverse (reductive) reaction using 3-keto-levoglucosan and NADH exhibited significantly lower and higher values than those of the forward reaction. The crystal structures of LGDH in the apo and complex forms with NADH, NADH + levoglucosan, and NADH + l-sorbose revealed that LGDH has a typical fold of Gfo/Idh/MocA family proteins, similar to those of -inositol dehydrogenase, aldose-aldose oxidoreductase, 1,5-anhydro-d-fructose reductase, and glucose-fructose oxidoreductase. The crystal structures also disclosed that the active site of LGDH is distinct from those of these enzymes. The LGDH active site extensively recognized the levoglucosan molecule with six hydrogen bonds, and the C3 atom of levoglucosan was closely located to the C4 atom of NADH nicotinamide. Our study is the first molecular characterization of LGDH, providing evidence for C3-specific oxidation and representing a starting point for future biotechnological use of LGDH and levoglucosan-metabolizing bacteria.
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http://dx.doi.org/10.1074/jbc.RA118.004963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231136PMC
November 2018

Structural features of a bacterial cyclic α-maltosyl-(1→6)-maltose (CMM) hydrolase critical for CMM recognition and hydrolysis.

J Biol Chem 2018 10 4;293(43):16874-16888. Epub 2018 Sep 4.

From the Department of Biotechnology and

Cyclic α-maltosyl-(1→6)-maltose (CMM, -{→6)-α-d-Glc-(1→4)-α-d-Glc-(1→6)-α-d-Glc-(1→4)-α-d-Glc-(1→})is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. CMM is composed of two maltose units and is one of the smallest cyclic glucooligosaccharides. Although CMM is resistant to usual amylases, it is efficiently hydrolyzed by CMM hydrolase (CMMase), belonging to subfamily 20 of glycoside hydrolase family 13 (GH13_20). Here, we determined the ligand-free crystal structure of CMMase from the soil-associated bacterium and its structures in complex with maltose, panose, and CMM to elucidate the structural basis of substrate recognition by CMMase. The structures disclosed that although the monomer structure consists of three domains commonly adopted by GH13 and other α-amylase-related enzymes, CMMase forms a unique wing-like dimer structure. The complex structure with CMM revealed four specific subsites, namely -3', -2, -1, and +1'. We also observed that the bound CMM molecule adopts a low-energy conformer compared with the X-ray structure of a single CMM crystal, also determined here. Comparison of the CMMase active site with those in other enzymes of the GH13_20 family revealed that three regions forming the wall of the cleft, denoted PYF (Pro-203/Tyr-204/Phe-205), CS (Cys-163/Ser-164), and Y (Tyr-168), are present only in CMMase and are involved in CMM recognition. Combinations of multiple substitutions in these regions markedly decreased the activity toward CMM, indicating that the specificity for this cyclic tetrasaccharide is supported by the entire shape of the pocket. In summary, our work uncovers the mechanistic basis for the highly specific interactions of CMMase with its substrate CMM.
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http://dx.doi.org/10.1074/jbc.RA118.004472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204909PMC
October 2018

Identification and characterization of a novel β-D-galactosidase that releases pyruvylated galactose.

Sci Rep 2018 08 13;8(1):12013. Epub 2018 Aug 13.

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan.

Pyruvyl modification of oligosaccharides is widely seen in both prokaryotes and eukaryotes. Although the biosynthetic mechanisms of pyruvylation have been investigated, enzymes that metabolize and degrade pyruvylated oligosaccharides are not well known. Here, we searched for a pyruvylated galactose (PvGal)-releasing enzyme by screening soil samples. We identified a Bacillus strain, as confirmed by the 16S ribosomal RNA gene analysis, that exhibited PvGal-ase activity toward p-nitrophenyl-β-D-pyruvylated galactopyranose (pNP-β-D-PvGal). Draft genome sequencing of this strain, named HMA207, identified three candidate genes encoding potential PvGal-ases, among which only the recombinant protein encoded by ORF1119 exhibited PvGal-ase activity. Although ORF1119 protein displayed broad substrate specificity for pNP sugars, pNP-β-D-PvGal was the most favorable substrate. The optimum pH for the ORF1119 PvGal-ase was determined as 7.5. A BLAST search suggested that ORF1119 homologs exist widely in bacteria. Among two homologs tested, BglC from Clostridium but not BglH from Bacillus showed PvGal-ase activity. Crystal structural analysis together with point mutation analysis revealed crucial amino acids for PvGal-ase activity. Moreover, ORF1119 protein catalyzed the hydrolysis of PvGal from galactomannan of Schizosaccharomyces pombe, suggesting that natural polysaccharides might be substrates of the PvGal-ase. This novel PvGal-catalyzing enzyme might be useful for glycoengineering projects to produce new oligosaccharide structures.
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http://dx.doi.org/10.1038/s41598-018-30508-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090015PMC
August 2018

Conformations of the type-1 lacto-N-biose I unit in protein complex structures.

Authors:
Shinya Fushinobu

Acta Crystallogr F Struct Biol Commun 2018 Aug 26;74(Pt 8):473-479. Epub 2018 Jul 26.

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

The lacto-N-biose I (Galβ1-3GlcNAc; LNB) disaccharide is present as a core unit of type-1 blood group antigens of animal glycoconjugates and milk oligosaccharides. Type-1 antigens often serve as cell-surface receptors for infection by pathogens. LNB in human milk oligosaccharides functions as a prebiotic for bifidobacteria and plays a key role in the symbiotic relationship of commensal gut microbes in infants. Protein Data Bank (PDB) entries exhibiting the LNB unit were investigated using the GlycoMapsDB web tool. There are currently 159 β-LNB and nine α-LNB moieties represented in ligands in the database. β-LNB and α-LNB moieties occur in 74 and six PDB entries, respectively, as NCS copies. The protein and enzyme structures are from various organisms including humans (galectins), viruses (haemagglutinin and capsid proteins), a pathogenic fungus, a parasitic nematode and protist, pathogenic bacteria (adhesins) and a symbiotic bacterium (a solute-binding protein of an ABC transporter). The conformations of LNB-containing glycans in enzymes vary significantly according to their mechanism of substrate recognition and catalysis. Analysis of glycosidic bond conformations indicated that the binding modes are significantly different in proteins adapted for modified or unmodified glycans.
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http://dx.doi.org/10.1107/S2053230X18006568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096478PMC
August 2018

Crystal structure of the central and the C-terminal RNase domains of colicin D implicated its translocation pathway through inner membrane of target cell.

J Biochem 2018 Nov;164(5):329-339

Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan.

Colicins are protein toxins produced by and toxic to Escherichia coli strains. Colicin D consists of an N-terminal domain (NTD), central domain (CD) and C-terminal RNase domain (CRD). The cognate immunity protein, ImmD, is co-synthesized in producer cells to block the toxic tRNase activity of the CRD. Previous studies have reported the crystal structure of CRD/ImmD complex. Colicin D hijacks the surface receptor FepA and the energy transducer TonB system using the NTD for translocation across the outer membrane of the target cells. The CD is required for endoproteolytic processing and the translocation of CRD across the inner membrane, and the membrane-associated protease FtsH and the signal peptidase LepB are exploited in this process. Although several regions of the CD have been identified in interactions with the hijacked inner membrane system or immunity protein, the structural basis of the CD is unknown. In this study, we determined the crystal structure of colicin D, containing both the CD and CRD. The full-length colicin D/ImmD heterodimer structure was built by superimposing the CD-CRD structure with the previously determined partial structures. The overall translocation process of colicin D, including the interaction between CD and LepB, is discussed.
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http://dx.doi.org/10.1093/jb/mvy056DOI Listing
November 2018

Purification, Cloning, Functional Expression, Structure, and Characterization of a Thermostable β-Mannanase from B168 and Its Efficiency in Production of Mannooligosaccharides from Coffee Wastes.

J Appl Glycosci (1999) 2018 20;65(2):13-21. Epub 2018 May 20.

2 Food Biotechnology Division, National Food Research Institute.

Highly thermostable β-mannanase, belonging to glycoside hydrolase family 5 subfamily 7, was purified from the culture supernatant of B168 and the cDNA of its transcript was cloned. The recombinant enzyme showed maximal activity at pH 4.5 and 85 °C. It retained more than 90 % of its activity below 60 °C. Obtaining the crystal structure of the enzyme helped us to understand the mechanism of its thermostability. An antiparallel β-sheet, salt-bridges, hydrophobic packing, proline residues in the loops, and loop shortening are considered to be related to the thermostability of the enzyme. The enzyme hydrolyzed mannans such as locust bean gum, carob galactomannan, guar gum, konjac glucomannan, and ivory nut mannan. It hydrolyzed 50.7 % of the total mannans from coffee waste, producing mannooligosaccharides. The enzyme has the highest optimum temperature among the known fungal β-mannanases and has potential for use in industrial applications.
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http://dx.doi.org/10.5458/jag.jag.JAG-2017_018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056896PMC
May 2018

Structural and thermodynamic insights into β-1,2-glucooligosaccharide capture by a solute-binding protein in .

J Biol Chem 2018 06 20;293(23):8812-8828. Epub 2018 Apr 20.

From the Department of Biotechnology,

β-1,2-Glucans are bacterial carbohydrates that exist in cyclic or linear forms and play an important role in infections and symbioses involving Gram-negative bacteria. Although several β-1,2-glucan-associated enzymes have been characterized, little is known about how β-1,2-glucan and its shorter oligosaccharides (Sop s) are captured and imported into the bacterial cell. Here, we report the biochemical and structural characteristics of the Sop -binding protein (SO-BP, Lin1841) associated with the ATP-binding cassette (ABC) transporter from the Gram-positive bacterium Calorimetric analysis revealed that SO-BP specifically binds to Sop s with a degree of polymerization of 3 or more, with values in the micromolar range. The crystal structures of SO-BP in an unliganded open form and in closed complexes with tri-, tetra-, and pentaoligosaccharides (Sop) were determined to a maximum resolution of 1.6 Å. The binding site displayed shape complementarity to Sop , which adopted a zigzag conformation. We noted that water-mediated hydrogen bonds and stacking interactions play a pivotal role in the recognition of Sop by SO-BP, consistent with its binding thermodynamics. Computational free-energy calculations and a mutational analysis confirmed that interactions with the third glucose moiety of Sop s are significantly responsible for ligand binding. A reduction in unfavorable changes in binding entropy that were in proportion to the lengths of the Sop s was explained by conformational entropy changes. Phylogenetic and sequence analyses indicated that SO-BP ABC transporter homologs, glycoside hydrolases, and other related proteins are co-localized in the genomes of several bacteria. This study may improve our understanding of bacterial β-1,2-glucan metabolism and promote the discovery of unidentified β-1,2-glucan-associated proteins.
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http://dx.doi.org/10.1074/jbc.RA117.001536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995521PMC
June 2018

Ligand complex structures of l-amino acid oxidase/monooxygenase from sp. AIU 813 and its conformational change.

FEBS Open Bio 2018 03 8;8(3):314-324. Epub 2018 Feb 8.

Department of Biotechnology The University of Tokyo Japan.

l-Amino acid oxidase/monooxygenase from sp. AIU 813 (l-AAO/MOG) catalyzes both the oxidative deamination and oxidative decarboxylation of the α-group of l-Lys to produce a keto acid and amide, respectively. l-AAO/MOG exhibits limited specificity for l-amino acid substrates with a basic side chain. We previously determined its ligand-free crystal structure and identified a key residue for maintaining the dual activities. Here, we determined the structures of l-AAO/MOG complexed with l-Lys, l-ornithine, and l-Arg and revealed its substrate recognition. Asp238 is located at the ceiling of a long hydrophobic pocket and forms a strong interaction with the terminal, positively charged group of the substrates. A mutational analysis on the D238A mutant indicated that the interaction is critical for substrate binding but not for catalytic control between the oxidase/monooxygenase activities. The catalytic activities of the D238E mutant unexpectedly increased, while the D238F mutant exhibited altered substrate specificity to long hydrophobic substrates. In the ligand-free structure, there are two channels connecting the active site and solvent, and a short region located at the dimer interface is disordered. In the l-Lys complex structure, a loop region is displaced to plug the channels. Moreover, the disordered region in the ligand-free structure forms a short helix in the substrate complex structures and creates the second binding site for the substrate. It is assumed that the amino acid substrate enters the active site of l-AAO/MOG through this route.

Database: The atomic coordinates and structure factors (codes 5YB6, 5YB7, and 5YB8) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Ec Numbers: 1.4.3.2 (l-amino acid oxidase), 1.13.12.2 (lysine 2-monooxygenase).
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http://dx.doi.org/10.1002/2211-5463.12387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832979PMC
March 2018

Discovery of α-l-arabinopyranosidases from human gut microbiome expands the diversity within glycoside hydrolase family 42.

J Biol Chem 2017 12 23;292(51):21092-21101. Epub 2017 Oct 23.

From the Department of Biotechnology, The University of Tokyo, Tokyo 113-8657, Japan,

Enzymes of the glycoside hydrolase family 42 (GH42) are widespread in bacteria of the human gut microbiome and play fundamental roles in the decomposition of both milk and plant oligosaccharides. All GH42 enzymes characterized so far have β-galactosidase activity. Here, we report the existence of a GH42 subfamily that is exclusively specific for α-l-arabinopyranoside and describe the first representative of this subfamily. We found that this enzyme (Arap42B) from a probiotic species cannot hydrolyze β-galactosides. However, Arap42B effectively hydrolyzed paeonolide and ginsenoside Rb2, plant glycosides containing an aromatic aglycone conjugated to α-l-arabinopyranosyl-(1,6)-β-d-glucopyranoside. Paeonolide, a natural glycoside from the roots of the plant genus is not hydrolyzed by classical GH42 β-galactosidases. X-ray crystallography revealed a unique Trp--Trp sequence motif at the Arap42B active site, as compared with a Phe--His motif in classical GH42 β-galactosidases. This analysis also indicated that the C6 position of galactose is blocked by the aromatic side chains, hence allowing accommodation only of Ara lacking this carbon. Automated docking of paeonolide revealed that it can fit into the Ara42B active site. The Glc moiety of paeonolide stacks onto the aromatic ring of the Trp at subsite +1 and C4-OH is hydrogen bonded with Asp Moreover, the aglycone stacks against Phe from the neighboring monomer in the Ara42B trimer, forming a proposed subsite +2. These results further support the notion that evolution of metabolic specialization can be tracked at the structural level in key enzymes facilitating degradation of specific glycans in an ecological niche.
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http://dx.doi.org/10.1074/jbc.M117.792598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743082PMC
December 2017

Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis.

Appl Environ Microbiol 2017 10 29;83(20). Epub 2017 Sep 29.

Department of Biotechnology, The University of Tokyo, Tokyo, Japan

Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from (AXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. AXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from indicated that AXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of lAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan. Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of thermoplastic polymers.
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http://dx.doi.org/10.1128/AEM.01251-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627002PMC
October 2017
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