Next-generation prebiotic promotes selective growth of bifidobacteria, suppressing Clostridioides difficile.

Certain existing prebiotics meant to facilitate the growth of beneficial bacteria in the intestine also promote the growth of other prominent bacteria. Therefore, the growth-promoting effects of ß-galactosides on intestinal bacteria were analyzed. Galactosyl-ß1,4-l-rhamnose (Gal-ß1,4-Rha) selectively promoted the growth of Bifidobacterium. Bifidobacterium longum subsp. longum 105-A (JCM 31944) has multiple solute-binding proteins belonging to ATP-binding cassette transporters for sugars. Each strain in the library of 11 B. longum subsp. longum mutants, in which each gene of the solute-binding protein was disrupted, was cultured in a medium containing Gal-ß1,4-Rha as the sole carbon source, and only the BL105A_0502 gene-disruption mutant showed delayed and reduced growth compared to the wild-type strain. BL105A_0502 homolog is highly conserved in bifidobacteria. In a Gal-ß1,4-Rha-containing medium, Bifidobacterium longum subsp. infantis JCM 1222T, which possesses BLIJ_2090, a homologous protein to BL105A_0502, suppressed the growth of enteric pathogen Clostridioides difficile, whereas the BLIJ_2090 gene-disrupted mutant did not. In vivo, administration of B. infantis and Gal-ß1,4-Rha alleviated C. difficile infection-related weight loss in mice. We have successfully screened Gal-ß1,4-Rha as a next-generation prebiotic candidate that specifically promotes the growth of beneficial bacteria without promoting the growth of prominent bacteria and pathogens.

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

Bifidobacterium longum is a symbiotic human gut bacterium that has a degradation system for ß-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 ß-linked arabinofuranosyl carbohydrates. We functionally and structurally analyzed a substrate-binding protein (SBP) of a putative ABC transporter (BLLJ_0208) in the ß-arabinooligosaccharide degradation system. Thermal shift assays and isothermal titration calorimetry revealed that the SBP specifically bound Araf-ß1,2-Araf (ß-Ara2 ) with a Kd of 0.150 µm, but did not bind L-arabinose or methyl-ß-Ara2 . Therefore, the SBP was termed ß-arabinobiose-binding protein (BABP). Crystal structures of BABP complexed with ß-Ara2 were determined at resolutions of up to 1.78 Å. The findings showed that ß-Ara2 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 ß-Ara2 , 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 ß-Ara2 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/).

Identification, characterization, and structural analyses of a fungal endo-ß-1,2-glucanase reveal a new glycoside hydrolase family.

endo-ß-1,2-Glucanase (SGL) is an enzyme that hydrolyzes ß-1,2-glucans, which play important physiological roles in some bacteria as a cyclic form. To date, no eukaryotic SGL has been identified. We purified an SGL from Talaromyces funiculosus (TfSGL), a soil fungus, to homogeneity and then cloned the complementary DNA encoding the enzyme. TfSGL shows no significant sequence similarity to any known glycoside hydrolase (GH) families, but shows significant similarity to certain eukaryotic proteins with unknown functions. The recombinant TfSGL (TfSGLr) specifically hydrolyzed linear and cyclic ß-1,2-glucans to sophorose (Glc-ß-1,2-Glc) as a main product. TfSGLr hydrolyzed reducing-end-modified ß-1,2-gluco-oligosaccharides to release a sophoroside with the modified moiety. These results indicate that TfSGL is an endo-type enzyme that preferably releases sophorose from the reducing end of substrates. Stereochemical analysis demonstrated that TfSGL is an inverting enzyme. The overall structure of TfSGLr includes an (α/α)6 toroid fold. The substrate-binding mode was revealed by the structure of a Michaelis complex of an inactive TfSGLr mutant with a ß-1,2-glucoheptasaccharide. Mutational analysis and action pattern analysis of ß-1,2-gluco-oligosaccharide derivatives revealed an unprecedented catalytic mechanism for substrate hydrolysis. Glu-262 (general acid) indirectly protonates the anomeric oxygen at subsite -1 via the 3-hydroxy group of the Glc moiety at subsite +2, and Asp-446 (general base) activates the nucleophilic water via another water. TfSGLr is apparently different from a GH144 SGL in the reaction and substrate recognition mechanism based on structural comparison. Overall, we propose that TfSGL and closely-related enzymes can be classified into a new family, GH162.

Characterization and Structural Analysis of a Novel exo-Type Enzyme Acting on ß-1,2-Glucooligosaccharides from Parabacteroides distasonis.

ß-1,2-Glucan is a polysaccharide produced mainly by some Gram-negative bacteria as a symbiosis and infectious factor. We recently identified endo-ß-1,2-glucanase from Chitinophaga pinensis ( CpSGL) as an enzyme comprising a new family. Here, we report the characteristics and crystal structure of a CpSGL homologue from Parabacteroides distasonis, an intestinal bacterium (BDI_3064 protein), which exhibits distinctive properties of known ß-1,2-glucan-degrading enzymes. BDI_3064 hydrolyzed linear ß-1,2-glucan and ß-1,2-glucooligosaccharides with degrees of polymerization (DPs) of ≥4 to produce sophorose specifically but did not hydrolyze cyclic ß-1,2-glucan. This result indicates that BDI_3064 is a new exo-type enzyme. BDI_3064 also produced sophorose from ß-1,2-glucooligosaccharide analogues that have a modified reducing end, indicating that BDI_3064 acts on its substrates from the nonreducing end. The crystal structure showed that BDI_3064 possesses additional N-terminal domains 1 and 2, unlike CpSGL. Superimposition of BDI_3064 and CpSGL complexed with ligands showed that R93 in domain 1 overlapped subsite -3 in CpSGL. Docking analysis involving a ß-1,2-glucooligosaccharide with DP4 showed that R93 completely blocks the nonreducing end of the docked ß-1,2-glucooligosaccharide. This indicates that BDI_3064 employs a distinct mechanism of recognition at the nonreducing end of substrates to act as an exo-type enzyme. Thus, we propose 2-ß-d-glucooligosaccharide sophorohydrolase (nonreducing end) as a systematic name for BDI_3064.

Function and structure relationships of a ß-1,2-glucooligosaccharide-degrading ß-glucosidase.

BT_3567 protein, a putative ß-glucosidase from Bacteroides thetaiotaomicron, exhibits higher activity toward Sop3-5 (Sopn , n: degree of polymerization of ß-1,2-glucooligosaccharides) than toward Sop2 , unlike a known ß-glucosidase from Listeria innocua which predominantly prefers Sop2 . In the complex structure determined by soaking of a D286N mutant crystal with Sop4 , a Sop3 moiety was observed at subsites -1 to +2. The glucose moiety at subsite +2 forms a hydrogen bond with Asn81, which is replaced with Gly in the L. innocua ß-glucosidase. The Km values of the N81G mutant for Sop3-5 are much higher than those of the wild-type, suggesting that Asn81 contributes to the binding to substrates longer than Sop3 .

Biochemical and structural analyses of a bacterial endo-ß-1,2-glucanase reveal a new glycoside hydrolase family.

ß-1,2-Glucan is an extracellular cyclic or linear polysaccharide from Gram-negative bacteria, with important roles in infection and symbiosis. Despite ß-1,2-glucan's importance in bacterial persistence and pathogenesis, only a few reports exist on enzymes acting on both cyclic and linear ß-1,2-glucan. To this end, we purified an endo-ß-1,2-glucanase to homogeneity from cell extracts of the environmental species Chitinophaga arvensicola, and an endo-ß-1,2-glucanase candidate gene (Cpin_6279) was cloned from the related species Chitinophaga pinensis The Cpin_6279 protein specifically hydrolyzed linear ß-1,2-glucan with polymerization degrees of ≥5 and a cyclic counterpart, indicating that Cpin_6279 is an endo-ß-1,2-glucananase. Stereochemical analysis demonstrated that the Cpin_6279-catalyzed reaction proceeds via an inverting mechanism. Cpin_6279 exhibited no significant sequence similarity with known glycoside hydrolases (GHs), and thus the enzyme defines a novel GH family, GH144. The crystal structures of the ligand-free and complex forms of Cpin_6279 with glucose (Glc) and sophorotriose (Glc-ß-1,2-Glc-ß-1,2-Glc) determined up to 1.7 Å revealed that it has a large cavity appropriate for polysaccharide degradation and adopts an (α/α)6-fold slightly similar to that of GH family 15 and 8 enzymes. Mutational analysis indicated that some of the highly conserved acidic residues in the active site are important for catalysis, and the Cpin_6279 active-site architecture provided insights into the substrate recognition by the enzyme. The biochemical characterization and crystal structure of this novel GH may enable discovery of other ß-1,2-glucanases and represent a critical advance toward elucidating structure-function relationships of GH enzymes.

Mechanistic insight into the substrate specificity of 1,2-ß-oligoglucan phosphorylase from Lachnoclostridium phytofermentans.

Glycoside phosphorylases catalyze the phosphorolysis of oligosaccharides into sugar phosphates. Recently, we found a novel phosphorylase acting on ß-1,2-glucooligosaccharides with degrees of polymerization of 3 or more (1,2-ß-oligoglucan phosphorylase, SOGP) in glycoside hydrolase family (GH) 94. Here, we characterized SOGP from Lachnoclostridium phytofermentans (LpSOGP) and determined its crystal structure. LpSOGP is a monomeric enzyme that contains a unique ß-sandwich domain (Ndom1) at its N-terminus. Unlike the dimeric GH94 enzymes possessing catalytic pockets at their dimer interface, LpSOGP has a catalytic pocket between Ndom1 and the catalytic domain. In the complex structure of LpSOGP with sophorose, sophorose binds at subsites +1 to +2. Notably, the Glc moiety at subsite +1 is flipped compared with the corresponding ligands in other GH94 enzymes. This inversion suggests the great distortion of the glycosidic bond between subsites -1 and +1, which is likely unfavorable for substrate binding. Compensation for this disadvantage at subsite +2 can be accounted for by the small distortion of the glycosidic bond in the sophorose molecule. Therefore, the binding mode at subsites +1 and +2 defines the substrate specificity of LpSOGP, which provides mechanistic insights into the substrate specificity of a phosphorylase acting on ß-1,2-glucooligosaccharides.

Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris.

BACKGROUND: Random mutagenesis is a powerful technique to obtain mutant proteins with different properties from the wild-type molecule. Error-prone PCR is often employed for random mutagenesis in bacterial protein expression systems, but has rarely been used in the methylotrophic yeast Pichia pastoris system, despite its significant advantages, mainly because large (µg-level) amounts of plasmids are required for transformation. RESULTS: We developed a quick and easy technique for random mutagenesis in P. pastoris by sequential Phi29 DNA polymerase-based amplification methods, error-prone rolling circle amplification (RCA) and multiple displacement amplification (MDA). The methodology was validated by applying it for random mutation of the gene encoding cellulase from the basidiomycete Phanerochaete chrysosporium (PcCel6A), a key enzyme in degradation of cellulosic biomass. In the error-prone RCA step, the concentrations of manganese ion (Mn(2+)) and cellulase gene-containing plasmid were varied, and the products obtained under each condition were subjected to the second MDA step in the absence of Mn(2+). The maximum error rate was 2.6 mutations/kb, as evaluated from the results of large-scale sequencing. Several µg of MDA products was transformed by electroporation into Pichia cells, and the activities of extracellularly expressed PcCel6A mutants towards crystalline and amorphous celluloses were compared with those of wild-type enzyme to identify key amino acid residues affecting degradation of crystalline cellulose. CONCLUSIONS: We present a rapid and convenient random mutagenesis method that does not require laborious steps such as ligation, cloning, and synthesis of specific primers. This method was successfully applied to the protein expression system in P. pastoris.

Functional and Structural Analysis of a ß-Glucosidase Involved in ß-1,2-Glucan Metabolism in Listeria innocua.

Despite the presence of ß-1,2-glucan in nature, few ß-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-ß-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 ß-glucosidase, we hypothesized that Lin1840 is also involved in ß-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-ß-1,2-Glc) among ß-1,2-glucooligosaccharides, suggesting that Lin1840 is a ß-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (ß-1,3), but not cellobiose (ß-1,4) or gentiobiose (ß-1,6) among ß-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-ß-1,2-Glc-ß-1,2-Glc) in spite of the higher Km value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into ß-1,2-glucooligosaccharide recognition by ß-glucosidase.

Discovery of a eukaryotic pyrroloquinoline quinone-dependent oxidoreductase belonging to a new auxiliary activity family in the database of carbohydrate-active enzymes.

Pyrroloquinoline quinone (PQQ) is a redox cofactor utilized by a number of prokaryotic dehydrogenases. Not all prokaryotic organisms are capable of synthesizing PQQ, even though it plays important roles in the growth and development of many organisms, including humans. The existence of PQQ-dependent enzymes in eukaryotes has been suggested based on homology studies or the presence of PQQ-binding motifs, but there has been no evidence that such enzymes utilize PQQ as a redox cofactor. However, during our studies of hemoproteins, we fortuitously discovered a novel PQQ-dependent sugar oxidoreductase in a mushroom, the basidiomycete Coprinopsis cinerea. The enzyme protein has a signal peptide for extracellular secretion and a domain for adsorption on cellulose, in addition to the PQQ-dependent sugar dehydrogenase and cytochrome domains. Although this enzyme shows low amino acid sequence homology with known PQQ-dependent enzymes, it strongly binds PQQ and shows PQQ-dependent activity. BLAST search uncovered the existence of many genes encoding homologous proteins in bacteria, archaea, amoebozoa, and fungi, and phylogenetic analysis suggested that these quinoproteins may be members of a new family that is widely distributed not only in prokaryotes, but also in eukaryotes.

Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII.

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Iα and IIII were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose Iα and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Iα and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Iα and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Iα and IIII and similar fractions of productive binding on cellulose Iα and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Iα and IIII with similar translational rates. With structural models of cellulose Iα and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes.

Directed evolution for thermostabilization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus.

To obtain a selection marker gene functional in a thermophilic bacterium, Thermus thermophilus, an in vivo-directed evolutionary strategy was conducted on a hygromycin B phosphotransferase gene (hyg) from Streptomyces hygroscopicus. The expression of wild-type hyg in T. thermophilus provided hygromycin B (HygB) resistance up to 60 °C. Through selection of mutants showing HygB resistance at higher temperatures, eight amino acid substitutions and the duplication of three amino acids were identified. A variant containing seven substitutions and the duplication (HYG10) showed HygB resistance at a highest temperature of 74 °C. Biochemical and biophysical analyses of recombinant HYG and HYG10 revealed that HYG10 was in fact thermostabilized. Modeling of the three-dimensional structure of HYG10 suggests the possible roles of the various substitutions and the duplication on thermostabilization, of which three substitutions and the duplication located at the enzyme surface suggested that these mutations made the enzyme more hydrophilic and provided increased stability in aqueous solution.

Adsorption characteristics of fungal family 1 cellulose-binding domain from Trichoderma reesei cellobiohydrolase I on crystalline cellulose: negative cooperative adsorption via a steric exclusion effect.

Cellobiohydrolases (CBHs) hydrolyzing crystalline cellulose share a two-domain structure of catalytic domain (CD) and cellulose-binding domain (CBD). To focus on the binding characteristics of CBD, we analyzed the adsorption of fusion protein of fungal family 1 CBD from Trichoderma reesei CBH I and red-fluorescent protein on crystalline and amorphous celluloses. Binding data were better fitted by Hill's model with negative cooperativity than by other adsorption models, suggesting the occurrence of a steric exclusion effect among the fusion molecules on the cellulose surfaces. The degree of negative cooperativity depended on the nature of the cellulose. The significance of this phenomenon for catalysis by intact CBHI is discussed.

Cellulose affinity purification of fusion proteins tagged with fungal family 1 cellulose-binding domain.

N- or C-terminal fusions of red-fluorescent protein (RFP) with various fungal cellulose-binding domains (CBDs) belonging to carbohydrate binding module (CBM) family 1 were expressed in a Pichia pastoris expression system, and the resulting fusion proteins were used to examine the feasibility of large-scale affinity purification of CBD-tagged proteins on cellulose columns. We found that RFP fused with CBD from Trichoderma reesei CBHI (CBD(Tr)(CBHI)) was expressed at up to 1.2g/l in the culture filtrate, which could be directly injected into the cellulose column. The fusion protein was tightly adsorbed on the cellulose column in the presence of a sufficient amount of ammonium sulfate and was efficiently eluted with pure water. Bovine serum albumin (BSA) was not captured under these conditions, whereas both BSA and the fusion protein were adsorbed on a phenyl column, indicating that the cellulose column can be used for the purification of not only hydrophilic proteins but also for hydrophobic proteins. Recovery of various fusion proteins exceeded 80%. Our results indicate that protein purification by expression of a target protein as a fusion with a fungal family 1 CBD tag in a yeast expression system, followed by affinity purification on a cellulose column, is simple, effective and easily scalable.

Enzymatic analysis of a thermostabilized mutant of an Escherichia coli hygromycin B phosphotransferase.

An Escherichia coli hygromycin B phosphotransferase (HPH) and its thermostabilized mutant protein, HPH5, containing five amino acid substitutions, D20G, A118V, S225P, Q226L, and T246A (Nakamura et al., J. Biosci. Bioeng., 100, 158-163 (2005)), obtained by an in vivo directed evolution procedure in Thermus thermophilus, were produced and purified from E. coli recombinants, and enzymatic comparisons were performed. The optimum temperatures for enzyme activity were 50 and 55 degrees C for HPH and HPH5 respectively, but the thermal stability of the enzyme activity and the temperature for protein denaturation of HPH5 increased, from 36 and 37.2 degrees C of HPH to 53 and 58.8 degrees C respectively. Specific activities and steady-state kinetics measured at 25 degrees C showed only slight differences between the two enzymes. From these results we concluded that HPH5 was thermostabilized at the protein level, and that the mutations introduced did not affect its enzyme activity, at least under the assay conditions.