Biology subjects

Yamada, C.

Publications and source records attributed to Yamada, C..

8 recordsLinked to original sources

bioRxiv2026

Crystal structure of a class II UDP-glucose-hexose-1-phosphate uridylyltransferase from Bifidobacterium longum involved in human milk oligosaccharide metabolism

Infant-associated bifidobacteria utilize lacto-N-biose I (LNB) and galacto-N-biose (GNB), major components of human milk oligosaccharides and intestinal mucin, respectively, through the GNB/LNB metabolic pathway. A UDP-glucose-hexose-1-phosphate uridylyltransferase (GalT) from Bifidobacterium longum JCM 1217 (BlGalT2) catalyzes a key step in this pathway. BlGalT2 belongs to class II GalTs within the histidine triad (HIT) superfamily, a rare protein group distinct from canonical class I GalTs. Here, we determined the crystal structure of BlGalT2 at 2.25 [A] resolution, providing the first three-dimensional structure of a class II GalTs. BlGalT2 is a monomeric enzyme with an architecture distinct from canonical class I GalT, comprising characteristic HIT1 and HIT2 catalytic core subdomains and an extended auxiliary domain that forms a self-contained active site. Kinetic analysis revealed a strong preference for GalNAc-1P over Gal-1P, with a 50-fold lower Km for GalNAc-1P, consistent with a hydrophobic pocket accommodating the N-acetyl group. Metal analysis and activity measurements indicated that Zn2+ is required for full activity, and structural prediction suggested its coordination by residues including the first histidine of the HIT motif. AlphaFold3 prediction and mutational analysis identified residues involved in nucleotide-sugar recognition and supported a Ping-Pong reaction mechanism involving a covalent enzyme-uridine monophosphate intermediate. These findings reveal how class II GalTs have evolved a unique structural framework and substrate specificity for N-acetylated sugars. Moreover, the structure of BlGalT2 fills the final missing gap in bifidobacterial HMO metabolism and provides molecular insight into the adaptation of bifidobacteria to host-derived glycans.

biochemistry↗

bioRxiv2024

Crystal structure of plant γ-glutamyl peptidase 1 with dual roles in sulfur metabolism and implications for oxidative stress regulation

{gamma}-Glutamyl peptidase 1 (GGP1) plays a dual role in primary and secondary sulfur metabolism in Arabidopsis thaliana. During glutathione (GSH) turnover, GGP1 hydrolyzes the isopeptide bond of GSH to degrade the tripeptide into Glu and Cys-Gly. During glucosinolate and camalexin biosynthesis, GGP1 processes GSH conjugates, which have a large substituent at the thiol side chain, by hydrolyzing the same isopeptide bond of {gamma}-Glu. In the present study, we determined the crystal structures of the following GGP1 forms: ligand-free, Glu complex, covalent {gamma}-Glu intermediate, and disulfide-linked S-S inactive forms. The intermediate structure, in which {gamma}-Glu is covalently linked to the nucleophile C100, was trapped by mutating the catalytic His to Asn (H192N). In the Glu complex and {gamma}-Glu intermediate structures, Glu bound to the S1 subsite is extensively recognized by several hydrogen bonds. The substrate recognition of the Cys-Gly moiety at the S1 and S2 subsites was revealed by modeling GSH in the active site. Mutational analysis indicated that R206 plays an important role in substrate binding by forming a salt bridge with Gly at the S2 subsite. An open pocket is present beyond the thiol side chain of Cys in the S1 subsite, which contributed to the dual activity of GGP1 toward GSH and GSH conjugates. The S-S inactive structure was obtained by soaking GGP1 crystals in Cys-Gly, and the catalytic cysteine (C100) partially formed a disulfide bond with a neighboring C154 residue. The partial inactivation of GGP1 in the presence of a pro-oxidant (Cys-Gly) has revealed its possible role in oxidative stress regulation in Arabidopsis.

biochemistry↗

bioRxiv2024

Identification and structural characterization of a novel acetyl xylan esterase from Aspergillus oryzae

Acetyl xylan esterase plays a crucial role in the degradation of xylan, the major plant hemicellulose, by liberating acetic acid from the backbone polysaccharides. Acetyl xylan esterase B from Aspergillus oryzae, designated AoAXEB, was biochemically and structurally investigated. The AoAXEB-encoding gene with a native signal peptide was successfully expressed in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 8.0 and 30 {degrees}C, respectively, and was stable up to 35{degrees}C. The optimal substrate for hydrolysis by purified recombinant AoAXEB among a panel of -naphthyl esters was -naphthyl acetate. Recombinant AoAXEB catalyzes the release of acetic acid from wheat arabinoxylan. The release of acetic acid from wheat arabinoxylan increases synergistically with xylanase addition. No activity was detected using the methyl esters of ferulic, p-coumaric, caffeic, or sinapic acids. The crystal structures of AoAXEB in the apo and succinate complexes were determined at resolutions of 1.75 and 1.90 [A], respectively. Although AoAXEB has been classified in the Esterase_phb family in the ESTerases and alpha/beta-Hydrolase Enzymes and Relatives (ESTHER) database, its structural features partly resemble those of ferulic acid esterase in the FaeC family. Phylogenetic analysis also indicated that AoAXEB is located between the clades of the two families. Docking analysis provided a plausible binding mode for xylotriose substrates acetylated at the 2- or 3-hydroxy position. This study expands the repertoire of side chain-degrading enzymes required for complete plant biomass degradation.

biochemistry↗

bioRxiv2024

Structural basis for the strict substrate specificity of beta-D-galactofuranosidase from Streptomyces sp. JHA19

D-Galactofuranose (Galf) is widely distributed in polysaccharides and glycoconjugates of bacteria, filamentous fungi, and protozoa. The biosynthetic and degradation pathways of Galf in pathogens have attracted attention as potential targets for drug development. {beta}-D-Galactofuranosidase (Galf-ase) releases Galf from the non-reducing ends of glycans. Galf-ase activity is often exhibited by -L-arabinofuranosidases, which hydrolyze a similar substrate. Several Galf-specific Galf-ases that cleave only Galf and not L-arabinofuranose (Araf) have recently been identified in the glycoside hydrolase (GH) families 2, 5, and 43. However, the structural basis of how they discriminate the substrates is unknown. ORF1110, belonging to GH2, is the first identified Galf-specific Galf-ase isolated from Streptomyces sp. JHA19. Here, we solved the crystal structure of ORF1110 in complex with a mechanism-based potent inhibitor, D-iminogalactitol (Ki = 65 M). ORF1110 binds to the C5-C6 hydroxy groups of D-iminogalactitol with an extensive and integral hydrogen bond network. This result suggests that in the case of Araf, which lacks the C6 hydroxymethyl group, this network is not formed. The domain structure of ORF1110 is similar to that of {beta}-glucuronidases and {beta}-galactosidases, which belong to the same GH2 family and hydrolyze pyranose substrates. However, their active site structures were completely different. A predicted structure of the C-terminal Abf domain of ORF1110 was very similar to the carbohydrate-binding module family 42, which binds Araf, and pockets that may bind Galf were present.

biochemistry↗

bioRxiv2021

MTCL2 promotes asymmetric microtubule organizationby crosslinking microtubules on the Golgi membrane

The Golgi complex plays an active role in organizing asymmetric microtubule arrays essential for polarized vesicle transport. The coiled-coil protein MTCL1 stabilizes microtubules nucleated from the Golgi membrane. Here, we report an MTCL1 paralog, MTCL2, which preferentially acts on the perinuclear microtubules accumulated around the Golgi. MTCL2 associates with the Golgi membrane through the N-terminal coiled-coil region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown of MTCL2 significantly impaired microtubule accumulation around the Golgi as well as the compactness of the Golgi ribbon assembly structure. Given that MTCL2 forms parallel oligomers through homo-interaction of the central coiled-coil motifs, our results indicate that MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane. Results of in vitro wound healing assays further suggest that this function of MTCL2 enables integration of the centrosomal and Golgi-associated microtubules on the Golgi membrane, supporting directional migration. Additionally, the results demonstrated the involvement of CLASPs and giantin in mediating the Golgi association of MTCL2.

cell biology↗

bioRxiv2020

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

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 two Acidianus 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 Sulfurisphaera tokodaii (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 [A] 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 [A] 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 and the other two cysteines (Cys101 and Cys104) had a supportive role. The cryo-EM structure was determined at 2.24 [A] 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. Given the high resolution achieved in this study, StSOR was shown to be a good benchmark sample for cryo-EM measurements. HighlightsO_LISulfur oxygenase reductase (SOR) was biochemically and structurally characterized. C_LIO_LIHigh resolution structures of SOR were determined by crystallography and cryo-EM. C_LIO_LITwenty-four identical subunits of SOR form a hollow sphere. C_LIO_LICatalytic components exhibited different features in the crystal and cryo-EM structures. C_LI

biochemistry↗

bioRxiv2020

Butyrate producing Clostridiales utilize distinct human milk oligosaccharides correlating to early colonization and prevalence in the human gut

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 unveil the growth of Roseburia and Eubacterium members on human milk oligosaccharides (HMOs) using an unprecedented catabolic apparatus. The described HMO pathways and additional glycan utilization loci confer co-growth with Akkermansia muciniphilia via cross-feeding and access to mucin O-glycans. Strikingly, both, HMO and xylooligosaccharide pathways, were active simultaneously attesting an adaptation to a mixed HMO-solid food diet. Analyses of 4599 Roseburia genomes underscored the preponderance of HMO pathways and highlighted different HMO utilization phylotypes. Our revelations provide a possible rationale for the early establishment and resilience of butyrate producing Clostridiales and expand the role of specific HMOs in the assembly of the early life microbiota.

microbiology↗

bioRxiv2020

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

Enzymes acting on -L-arabinofuranosides have been extensively studied; however, the structures and functions of {beta}-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 {beta}-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular {beta}-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing {beta}-1,2-linked arabinofuranose disaccharide ({beta}-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 [A] resolution. The HypBA2 structure consists of a central catalytic (/)6 barrel domain and two flanking (N- and C-terminal) {beta}-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the {beta}-Ara2 disaccharide; this pocket is highly conserved among GH121 proteins. The three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all ~270 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 GH63 -glycosidase, GH94 chitobiose phosphorylase, GH142 {beta}-L-arabinofuranosidase, GH78 -L-rhamnosidase, and GH37 ,-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. {beta}-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 {beta}-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens.

biochemistry↗