Biology subjects

Kashima, T.

Publications and source records attributed to Kashima, T..

7 recordsLinked to original sources

bioRxiv2026

Structural mechanism defining product specificity in glycoside hydrolase family 66 cycloisomaltotetraose glucanotransferase

Cyclic oligosaccharides possess industrial advantages, including molecular encapsulation capability and high physicochemical stability, owing to the absence of a reducing end. Recently, a novel cyclic tetrasaccharide, cycloisomaltotetraose (CI4), consisting of four -1,6-linked glucose units, and the enzymes responsible for its synthesis, cycloisomaltotetraose glucanotransferases (CI4Tases), were discovered. Unlike known cycloisomaltooligosaccharide glucanotransferases (CITases) that yield a wide distribution of cyclic products with a degree of polymerization (DP) of 7 or higher, CI4Tases strictly produce CI4. To elucidate the molecular mechanism underlying this strict DP4 specificity, we determined the crystal structures of CI4Tase from Agreia sp. D1110, in its ligand-free form, as well as in complex with the linear hydrolysis product isomaltotetraose (IG4) and with CI4. Structural comparisons revealed that a loop (M247 to R251) blocks the region corresponding to the -5 subsite of typical CITases, narrowing the substrate-binding pocket. This "molecular ruler" mechanism ensures that only a glycan chain of exactly four glucose units is accommodated for cyclization. Among mutants of the residue positioned at the center of bound CI4, the formation of by-products other than CI4 was significantly suppressed in F245L, F245A, and F245W. While the cyclization activity of all F245 mutants decreased, the CI4 hydrolysis activity of these three mutants was also significantly reduced, resulting in an increased specificity for cyclic sugar production. These findings elucidate the strict size-control mechanism of CI4Tase and provide a structural foundation for engineering cycloisomaltooligosaccharide-producing enzymes with optimized transglycosylation efficiency and specificity for industrial applications.

biochemistry↗

bioRxiv2026

Reprogramming protein interfaces between adenylation and carrier protein domains in nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) assemble structurally diverse bioactive natural products through selective communication between adenylation (A) and carrier protein (CP) domains. Although rational rewiring of these protein-protein interactions could enable customized NRPS design, this remains challenging due to the dynamic nature of protein interfaces. Here we show that structure-guided interface reprogramming enables productive non-cognate A- CP pairings across enterobactin, vibriobactin, pyochelin, and vicenistatin biosynthetic systems. Engineered interactions between the A domains EntE, VibE, or PchD and the non-cognate CPs VinL or EntB were validated by biochemical, kinetic, and structural analyses. Reconstituted pathways containing engineered VibE, EntB, and EntF restored enterobactin production and increased yield to 2.2-fold that of the native EntE-EntB-EntF system. Interface-engineered PchD also enhanced production of a non-native salicylic acid-norspermidine conjugate. An X-ray structure and molecular dynamics simulations of an engineered non-cognate A-CP complex reveal recognition principles for programmable NRPS interface design.

biochemistry↗

bioRxiv2026

Discovery and structural analysis of glycoside hydrolase family 176 α-1,2 glucosidase from Arthrobacter humicola A8F5

Glycoside hydrolases (GHs) exhibit remarkable specificity dictated by the structural configuration of their target glycosidic linkages. While enzymes that process -1,4- and -1,6-linkages in starch or glycogen are well-characterized, those acting on less common bonds, such as -1,2-glucosidic linkages, remain largely underexplored. In this study, we report the discovery and structural elucidation of a novel -1,2-glucosidase from Arthrobacter humicola A8F5 (A8F5 glucosidase), representing a newly uncovered activity within the poorly characterized GH176 family. Biochemical characterizations revealed that A8F5 glucosidase exclusively cleaves -1,2-linkages via an anomer-inverting mechanism, with a distinct preference for short kojioligosaccharides. To circumvent crystallization obstacles caused by high loop flexibility and translational non-crystallographic symmetry, we engineered a loop-truncated variant. This strategy enabled the determination of high-resolution (up to 1.79 [A]) crystal structures of the enzyme in its ligand-free form and in complex with kojibiose, kojitriose, and selaginose. A8F5 glucosidase adopts a (/)6-barrel fold characteristic of clan GH-G. Complementing the crystal structures with AlphaFold3 prediction demonstrated that two prominent active-site loops (loops 3 and 4) adopt a closed conformation that constricts the catalytic pocket, rendering the architecture suitable for short oligosaccharide recognition while restricting access to larger polymers. Furthermore, sequence similarity network analysis highlights vast, uncharacterized functional diversity within the GH176 family. These findings revealed that the GH176 enzyme recognizes and hydrolyses -1,2-glucosidic bonds through a structural framework distinct from that of the previously known clan GH-L GH65 kojibiose hydrolase, expanding the known functional landscape of this enzyme group toward rare -glucans.

biochemistry↗

bioRxiv2025

Discovery of a wide variety of α-1,6-cyclized β-1,2-glucan synthases: a new entrance for host-microbe interactions.

-1,6-Cyclized {beta}-1,2-glucans (C{beta}G) are established virulence factors in Xanthomonas, yet functional breadth and distribution of C{beta}Gs in nature have remained unclear. Here, we biochemically identified enzymes synthesizing C{beta}Gs with specific chain lengths, exhibiting potential natural occurrence of various C{beta}Gs. Structural analyses identify subtle variations in a loop, named Loop X, as a key determinant of product size, leading to understanding of the whole picture of the mechanism that controls the sizes. We further demonstrated that C{beta}G composed of 13 glucose units contributes to plant-virulence in Ralstonia pseudosolanacearum, expanding the functional scope of virulence-associated cyclic glucans. Overall, it provides the potential targets for regulating various plant-microbe interactions and also serves as a vital lead to discovering unknown host-microbe interactions with significant potential for agricultural applications.

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

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↗

bioRxiv2024

Crystal structure of Bifidobacterium bifidum glycoside hydrolase family 110 α-galactosidase specific for blood group B antigen

To overcome incompatibility issues and increase the possibility of blood transfusion, technologies that enable efficient conversion of A- and B-type red blood cells to the universal donor O-type is desirable. Although several blood type-converting enzymes have been identified, detailed understanding about their molecular functions is limited. -Galactosidase from Bifidobacterium bifidum JCM 1254 (AgaBb), belonging to glycoside hydrolase (GH) 110 subfamily A, specifically acts on blood group B antigen. Here we present the crystal structure of AgaBb, including the catalytic GH110 domain and part of the C-terminal uncharacterized regions. Based on this structure, we deduced a possible binding mechanism of blood group B antigen to the active site. Site-directed mutagenesis confirmed that R270 and E380 recognize the fucose moiety in the B antigen. Thermal shift assay revealed that the C-terminal uncharacterized region significantly contributes to protein stability. This region is shared only among GH110 enzymes from B. bifidum and some Ruminococcus species. The elucidation of the molecular basis for the specific recognition of blood group B antigen is expected to lead to the practical application of blood group conversion enzymes in the future.

biochemistry↗