Bacteroidota polysaccharide utilization system for branched dextran exopolysaccharides from lactic acid bacteria.

Dextran is an α-(1→6)-glucan that is synthesized by some lactic acid bacteria, and branched dextran with α-(1→2)-, α-(1→3)-, and α-(1→4)-linkages are often produced. Although many dextranases are known to act on the α-(1→6)-linkage of dextran, few studies have functionally analyzed the proteins involved in degrading branched dextran. The mechanism by which bacteria utilize branched dextran is unknown. Earlier, we identified dextranase (FjDex31A) and kojibiose hydrolase (FjGH65A) in the dextran utilization locus (FjDexUL) of a soil Bacteroidota Flavobacterium johnsoniae and hypothesized that FjDexUL is involved in the degradation of α-(1→2)-branched dextran. In this study, we demonstrate that FjDexUL proteins recognize and degrade α-(1→2)- and α-(1→3)-branched dextrans produced by Leuconostoc citreum S-32 (S-32 α-glucan). The FjDexUL genes were significantly upregulated when S-32 α-glucan was the carbon source compared with α-glucooligosaccharides and α-glucans, such as linear dextran and branched α-glucan from L. citreum S-64. FjDexUL glycoside hydrolases synergistically degraded S-32 α-glucan. The crystal structure of FjGH66 shows that some sugar-binding subsites can accommodate α-(1→2)- and α-(1→3)-branches. The structure of FjGH65A in complex with isomaltose supports that FjGH65A acts on α-(1→2)-glucosyl isomaltooligosaccharides. Furthermore, two cell surface sugar-binding proteins (FjDusD and FjDusE) were characterized, and FjDusD showed an affinity for isomaltooligosaccharides and FjDusE for dextran, including linear and branched dextrans. Collectively, FjDexUL proteins are suggested to be involved in the degradation of α-(1→2)- and α-(1→3)-branched dextrans. Our results will be helpful in understanding the bacterial nutrient requirements and symbiotic relationships between bacteria at the molecular level.

Utilization of dietary mixed-linkage ß-glucans by the Firmicute Blautia producta.

The ß-glucans are structurally varied, naturally occurring components of the cell walls, and storage materials of a variety of plant and microbial species. In the human diet, mixed-linkage glucans [MLG - ß-(1,3/4)-glucans] influence the gut microbiome and the host immune system. Although consumed daily, the molecular mechanism by which human gut Gram-positive bacteria utilize MLG largely remains unknown. In this study, we used Blautia producta ATCC 27340 as a model organism to develop an understanding of MLG utilization. B. producta encodes a gene locus comprising a multi-modular cell-anchored endo-glucanase (BpGH16MLG), an ABC transporter, and a glycoside phosphorylase (BpGH94MLG) for utilizing MLG, as evidenced by the upregulation of expression of the enzyme- and solute binding protein (SBP)-encoding genes in this cluster when the organism is grown on MLG. We determined that recombinant BpGH16MLG cleaved various types of ß-glucan, generating oligosaccharides suitable for cellular uptake by B. producta. Cytoplasmic digestion of these oligosaccharides is then performed by recombinant BpGH94MLG and ß-glucosidases (BpGH3-AR8MLG and BpGH3-X62MLG). Using targeted deletion, we demonstrated BpSBPMLG is essential for B. producta growth on barley ß-glucan. Furthermore, we revealed that beneficial bacteria, such as Roseburia faecis JCM 17581T, Bifidobacterium pseudocatenulatum JCM 1200T, Bifidobacterium adolescentis JCM 1275T, and Bifidobacterium bifidum JCM 1254, can also utilize oligosaccharides resulting from the action of BpGH16MLG. Disentangling the ß-glucan utilizing the capability of B. producta provides a rational basis on which to consider the probiotic potential of this class of organism.

Structural basis for the recognition of α-1,6-branched α-glucan by GH13_47 α-amylase from Rhodothermus marinus.

Glycoside hydrolase (GH) family 13 is among the main families of enzymes acting on starch; recently, subfamily 47 of GH13 (GH13_47) has been established. The crystal structure and function of a GH13_47 enzyme from Bacteroides ovatus has only been reported to date. This enzyme has α-amylase activity, while the GH13_47 enzymes comprise approximately 800-900 amino acid residues which are almost double those of typical α-amylases. It is important to know how different the GH13_47 enzymes are from other α-amylases. Rhodothermus marinus JCM9785, a thermophilic bacterium, possesses a gene for the GH13_47 enzyme, which is designated here as RmGH13_47A. Its structure has been predicted to be composed of seven domains: N1, N2, N3, A, B, C, and D. We constructed a plasmid encoding Gly266-Glu886, which contains the N3, A, B, and C domains and expressed the protein in Escherichia coli. The enzyme hydrolyzed starch and pullulan by a neopullulanase-type action. Additionally, the enzyme acted on maltotetraose, and saccharides with α-1,6-glucosidic linkages were observed in the products. Following the replacement of the catalytic residue Asp563 with Ala, the crystal structure of the variant D563A in complex with the enzymatic products from maltotetraose was determined; as a result, electron density for an α-1,6-branched pentasaccharide was observed in the catalytic pocket, and Ile762 and Asp763 interacted with the branched chain of the pentasaccharide. These findings suggest that RmGH13_47A is an α-amylase that prefers α-1,6-branched parts of starch to produce oligosaccharides.

Structural basis of the strict specificity of a bacterial GH31 α-1,3-glucosidase for nigerooligosaccharides.

Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The kcat/Km values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members.

Structure of a bacterial α-1,2-glucosidase defines mechanisms of hydrolysis and substrate specificity in GH65 family hydrolases.

Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no crystal structure of a GH65 GH has yet been reported. In this study, we use biochemical experiments and X-ray crystallography to examine the function and structure of a GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) that shows low amino acid sequence homology to reported GH65 enzymes. We found that FjGH65A does not exhibit phosphorolytic activity, but it does hydrolyze kojibiose (α-1,2-glucobiose) and oligosaccharides containing a kojibiosyl moiety without requiring inorganic phosphate. In addition, stereochemical analysis demonstrated that FjGH65A catalyzes this hydrolytic reaction via an anomer-inverting mechanism. The three-dimensional structures of FjGH65A in native form and in complex with glucose were determined at resolutions of 1.54 and 1.40 Å resolutions, respectively. The overall structure of FjGH65A resembled those of other GH65 GPs, and the general acid catalyst Glu472 was conserved. However, the amino acid sequence forming the phosphate-binding site typical of GH65 GPs was not conserved in FjGH65A. Moreover, FjGH65A had the general base catalyst Glu616 instead, which is required to activate a nucleophilic water molecule. These results indicate that FjGH65A is an α-1,2-glucosidase and is the first bacterial GH found in the GH65 family.

Crystal structure and sugar-binding ability of the C-terminal domain of N-acetylglucosaminyltransferase IV establish a new carbohydrate-binding module family.

N-glycans are modified by glycosyltransferases in the endoplasmic reticulum and Golgi apparatus. N-acetylglucosaminyltransferase IV (GnT-IV) is a Golgi-localized glycosyltransferase that synthesizes complex-type N-glycans in vertebrates. This enzyme attaches N-acetylglucosamine (GlcNAc) to the α-1,3-linked mannose branch of the N-glycan core structure via a ß-1,4 linkage. Deficiency of this enzyme is known to cause abnormal cellular functions, making it a vital enzyme for living organisms. However, there has been no report on its 3-dimensional structure to date. Here, we demonstrated that the C-terminal regions (named CBML) of human GnT-IVa and Bombyx mori ortholog have the ability to bind ß-N-acetylglucosamine. In addition, we determined the crystal structures of human CBML, B. mori CBML, and its complex with ß-GlcNAc at 1.97, 1.47, and 1.15 Å resolutions, respectively, and showed that they adopt a ß-sandwich fold, similar to carbohydrate-binding module family 32 (CBM32) proteins. The regions homologous to CBML (≥24% identity) were found in GnT-IV isozymes, GnT-IVb, and GnT-IVc (known as GnT-VI), and the structure of B. mori CBML in complex with ß-GlcNAc indicated that the GlcNAc-binding residues were highly conserved among these isozymes. These residues are also conserved with the GlcNAc-binding CBM32 domain of ß-N-acetylhexosaminidase NagH from Clostridium perfringens despite the low sequence identity (<20%). Taken together with the phylogenetic analysis, these findings indicate that these CBMLs may be novel CBM family proteins with GlcNAc-binding ability.

Unlocking the Hydrolytic Mechanism of GH92 α-1,2-Mannosidases: Computation Inspires the use of C-Glycosides as Michaelis Complex Mimics.

The conformational changes in a sugar moiety along the hydrolytic pathway are key to understand the mechanism of glycoside hydrolases (GHs) and to design new inhibitors. The two predominant itineraries for mannosidases go via O S2 →B2,5 →1 S5 and 3 S1 →3 H4 →1 C4 . For the CAZy family 92, the conformational itinerary was unknown. Published complexes of Bacteroides thetaiotaomicron GH92 catalyst with a S-glycoside and mannoimidazole indicate a 4 C1 →4 H5 /1 S5 →1 S5 mechanism. However, as observed with the GH125 family, S-glycosides may not act always as good mimics of GH's natural substrate. Here we present a cooperative study between computations and experiments where our results predict the E5 →B2,5 /1 S5 →1 S5 pathway for GH92 enzymes. Furthermore, we demonstrate the Michaelis complex mimicry of a new kind of C-disaccharides, whose biochemical applicability was still a chimera.

Rational conversion of chromophore selectivity of cyanobacteriochromes to accept mammalian intrinsic biliverdin.

Because cyanobacteriochrome photoreceptors need only a single compact domain for chromophore incorporation and for absorption of visible spectra including the long-wavelength far-red region, these molecules have been paid much attention for application to bioimaging and optogenetics. Most cyanobacteriochromes, however, have a drawback to incorporate phycocyanobilin that is not available in the mammalian cells. In this study, we focused on biliverdin (BV) that is a mammalian intrinsic chromophore and absorbs the far-red region and revealed that replacement of only four residues was enough for conversion from BV-rejective cyanobacteriochromes into BV-acceptable molecules. We succeeded in determining the crystal structure of one of such engineered molecules, AnPixJg2_BV4, at 1.6 Å resolution. This structure identified unusual covalent bond linkage, which resulted in deep BV insertion into the protein pocket. The four mutated residues contributed to reducing steric hindrances derived from the deeper insertion. We introduced these residues into other domains, and one of them, NpF2164g5_BV4, produced bright near-infrared fluorescence from mammalian liver in vivo. Collectively, this study provides not only molecular basis to incorporate BV by the cyanobacteriochromes but also rational strategy to open the door for application of cyanobacteriochromes to visualization and regulation of deep mammalian tissues.

Structure-function analysis of silkworm sucrose hydrolase uncovers the mechanism of substrate specificity in GH13 subfamily 17 exo-α-glucosidases.

The domestic silkworm Bombyx mori expresses two sucrose-hydrolyzing enzymes, BmSUH and BmSUC1, belonging to glycoside hydrolase family 13 subfamily 17 (GH13_17) and GH32, respectively. BmSUH has little activity on maltooligosaccharides, whereas other insect GH13_17 α-glucosidases are active on sucrose and maltooligosaccharides. Little is currently known about the structural mechanisms and substrate specificity of GH13_17 enzymes. In this study, we examined the crystal structures of BmSUH without ligands; in complexes with substrates, products, and inhibitors; and complexed with its covalent intermediate at 1.60-1.85 Å resolutions. These structures revealed that the conformations of amino acid residues around subsite -1 are notably different at each step of the hydrolytic reaction. Such changes have not been previously reported among GH13 enzymes, including exo- and endo-acting hydrolases, such as α-glucosidases and α-amylases. Amino acid residues at subsite +1 are not conserved in BmSUH and other GH13_17 α-glucosidases, but subsite -1 residues are absolutely conserved. Substitutions in three subsite +1 residues, Gln191, Tyr251, and Glu440, decreased sucrose hydrolysis and increased maltase activity of BmSUH, indicating that these residues are key for determining its substrate specificity. These results provide detailed insights into structure-function relationships in GH13 enzymes and into the molecular evolution of insect GH13_17 α-glucosidases.

G-quadruplex binding ability of TLS/FUS depends on the ß-spiral structure of the RGG domain.

The RGG domain, defined as closely spaced Arg-Gly-Gly repeats, is a DNA and RNA-binding domain in various nucleic acid-binding proteins. Translocated in liposarcoma (TLS), which is also called FUS, is a protein with three RGG domains, RGG1, RGG2 and RGG3. TLS/FUS binding to G-quadruplex telomere DNA and telomeric repeat-containing RNA depends especially on RGG3, comprising Arg-Gly-Gly repeats with proline- and arginine-rich regions. So far, however, only non-specific DNA and RNA binding of TLS/FUS purified with buffers containing urea and KCl have been reported. Here, we demonstrate that protein purification using a buffer with high concentrations of urea and KCl decreases the G-quadruplex binding abilities of TLS/FUS and RGG3, and disrupts the ß-spiral structure of RGG3. Moreover, the Arg-Gly-Gly repeat region in RGG3 by itself cannot form a stable ß-spiral structure that binds to the G-quadruplex, because the proline- and arginine-rich regions induce the ß-spiral structure and the G-quadruplex-binding ability of RGG3. Our findings suggest that the G-quadruplex-specific binding abilities of TLS/FUS require RGG3 with a ß-spiral structure stabilized by adjacent proline- and arginine-regions.

A novel intracellular dextranase derived from Paenibacillus sp. 598K with an ability to degrade cycloisomaltooligosaccharides.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (CIs) in culture from dextran and starch. CIs are cyclic oligosaccharides consisting of seven or more α-(1 → 6)-linked-D-glucose residues. The extracellular enzyme CI glucanotransferase (PsCITase), which is the member of glycoside hydrolase family 66, catalyzes the final stage of CI production and produces mainly cycloisomaltoheptaose. We have discovered a novel intracellular CI-degrading dextranase (PsDEX598) from Paenibacillus sp. 598K. The 69.7-kDa recombinant PsDEX598 does not digest isomaltotetraose or shorter isomaltooligosaccharides, but digests longer ones of at least up to isomaltoheptaose. It also digests oligoCIs of cycloisomaltoheptaose, cycloisomaltooctaose, and cycloisomaltononaose better than it does with megaloCIs of cycloisomaltodecaose, cycloisomaltoundecaose, and cycloisomaltododecaose, as well as an α-(1 → 6)-D-glucan of dextran 40. PsDEX598 is produced intracellularly when culture medium is supplemented with cycloisomaltoheptaose or dextran, but not with isomaltooligosaccharides (a mixture of isomaltose, isomaltotriose, and panose), starch, or glucose. The whole genomic DNA sequence of the strain 598K implies that it harbors two genes for enzymes belonging to glycoside hydrolase family 66 (PsCITase and PsDEX598), and PsDEX598 is the only dextranase in the strain. PsDEX598 does not have any carbohydrate-binding modules (CBMs) and has a low similarity (< 30%) with other family 66 dextranases, and the catalytic amino acids of this enzyme are predicted to be Asp191, Asp303, and Glu368. The strain Paenibacillus sp. 598K appears to take up CI-7, so these findings indicate that this bacterium can degrade CIs using a dextranase within the cells and so utilize them as a carbon source for growth.

Combined Drug Resistance Mutations Substantially Enhance Enzyme Production in Paenibacillus agaridevorans.

This study shows that sequential introduction of drug resistance mutations substantially increased enzyme production in Paenibacillus agaridevorans The triple mutant YT478 (rsmG Gln225→stop codon, rpsL K56R, and rpoB R485H), generated by screening for resistance to streptomycin and rifampin, expressed a 1,100-fold-larger amount of the extracellular enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) than the wild-type strain. These mutants were characterized by higher intracellular S-adenosylmethionine concentrations during exponential phase and enhanced protein synthesis activity during stationary phase. Surprisingly, the maximal expression of CITase mRNA was similar in the wild-type and triple mutant strains, but the mutant showed greater CITase mRNA expression throughout the growth curve, resulting in enzyme overproduction. A metabolome analysis showed that the triple mutant YT478 had higher levels of nucleic acids and glycolysis metabolites than the wild type, indicating that YT478 mutant cells were activated. The production of CITase by the triple mutant was further enhanced by introducing a mutation conferring resistance to the rare earth element, scandium. This combined drug resistance mutation method also effectively enhanced the production of amylases, proteases, and agarases by P. agaridevorans and Streptomyces coelicolor This method also activated the silent or weak expression of the P. agaridevorans CITase gene, as shown by comparisons of the CITase gene loci of P. agaridevorans T-3040 and another cycloisomaltooligosaccharide-producing bacterium, Paenibacillus sp. strain 598K. The simplicity and wide applicability of this method should facilitate not only industrial enzyme production but also the identification of dormant enzymes by activating the expression of silent or weakly expressed genes.IMPORTANCE Enzyme use has become more widespread in industry. This study evaluated the molecular basis and effectiveness of ribosome engineering in markedly enhancing enzyme production (>1,000-fold). This method, due to its simplicity, wide applicability, and scalability for large-scale production, should facilitate not only industrial enzyme production but also the identification of novel enzymes, because microorganisms contain many silent or weakly expressed genes which encode novel antibiotics or enzymes. Furthermore, this study provides a new mechanism for strain improvement, with a consistent rather than transient high expression of the key gene(s) involved in enzyme production.

Expression of a functional intrabody against hepatitis C virus core protein in Escherichia coli and silkworm pupae.

It has been shown that the single-domain intrabody 2H9-L against the hepatitis C virus (HCV) capsid (core) protein inhibits the viral propagation and NF-κB promoter activity induced by the HCV core. In this study, 2H9-L fused with the FLAG tag sequence was expressed in both Escherichia coli and silkworm pupae and then purified. In addition, the full-length and its C terminal deletions of the HCV core protein, i.e., 1-123 amino acid residues (C123), 1-152 amino acid residues (C152), 1-177 amino acid residues (C177) and 1-191 amino acid residues (C191), were expressed as fusion proteins with a 6 × His tag at their N-terminus in E. coli and then purified. Approximately 175 and 132 µg of the intrabody were purified from 100 ml of E. coli culture and 10 silkworm pupae, respectively, by affinity chromatography. The C123, C152, C177 and C191 HCV core protein variants were purified to approximately 152, 127, 103 and 155 µg, respectively, from 100 ml of E. coli culture. An ELISA in which the intrabodies were immobilized revealed that the intrabodies purified from both hosts were bound to all HCV core protein variants. However, their binding to the C191 appeared to be weak compared to their bindings to the other HCV core protein variants. When C152 was immobilized in the ELISA, the binding of each intrabody to the core protein was also observed. These purified intrabodies can be used in biochemical analyses of the inhibitory mechanism of HCV propagation and as protein interference reagents, thus providing a potential pathway to developing a new type of antiviral drug.

Paenibacillus sp. 598K 6-α-glucosyltransferase is essential for cycloisomaltooligosaccharide synthesis from α-(1 → 4)-glucan.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (cyclodextrans) from starch even in the absence of dextran. Cycloisomaltooligosaccharide glucanotransferase synthesizes cycloisomaltooligosaccharides exclusively from an α-(1 â†’ 6)-consecutive glucose chain consisting of at least four molecules. Starch is not a substrate of this enzyme. Therefore, we predicted that the bacterium possesses another enzyme system for extending α-(1 â†’ 6)-linked glucoses from starch, which can be used as the substrate for cycloisomaltooligosaccharide glucanotransferase, and identified the transglucosylation enzyme Ps6GT31A. We purified Ps6GT31A from the bacterial culture supernatant, cloned its corresponding gene, and characterized the recombinant enzyme. Ps6GT31A belongs to glycoside hydrolase family 31, and it liberates glucose from the non-reducing end of the substrate in the following order of activity: α-(1 â†’ 4)-> α-(1 â†’ 2)- > α-(1 â†’ 3)- > α-(1 â†’ 6)-glucobiose and maltopentaose > maltotetraose > maltotriose > maltose. Ps6GT31A catalyzes both hydrolysis and transglucosylation. The resulting transglucosylation compounds were analyzed by high-performance liquid chromatography and mass spectrometry. Analysis of the initial products by 13C nuclear magnetic resonance spectroscopy revealed that Ps6GT31A had a strong α-(1 â†’ 4) to α-(1 â†’ 6) transglucosylation activity. Ps6GT31A elongated α-(1 â†’ 6)-linked glucooligosaccharide to at least a degree of polymerization of 10 through a successive transglucosylation reaction. Eventually, cycloisomaltooligosaccharide glucanotransferase creates cycloisomaltooligosaccharides using the transglucosylation products generated by Ps6GT31A as the substrates. Our data suggest that Ps6GT31A is the key enzyme to synthesize α-(1 â†’ 6)-glucan for cycloisomaltooligosaccharide production in dextran-free environments.

Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by family 63 inverting α-glycosidase.

Glycoside hydrolases are divided into two groups, known as inverting and retaining enzymes, based on their hydrolytic mechanisms. Glycoside hydrolase family 63 (GH63) is composed of inverting α-glycosidases, which act mainly on α-glucosides. We previously found that Escherichia coli GH63 enzyme, YgjK, can hydrolyze 2-O-α-d-glucosyl-d-galactose. Two constructed glycosynthase mutants, D324N and E727A, which catalyze the transfer of a ß-glucosyl fluoride donor to galactose, lactose, and melibiose. Here, we determined the crystal structures of D324N and E727A soaked with a mixture of glucose and lactose at 1.8- and 2.1-Å resolutions, respectively. Because glucose and lactose molecules are found at the active sites in both structures, it is possible that these structures mimic the enzyme-product complex of YgjK. A glucose molecule found at subsite -1 in both structures adopts an unusual 1S3 skew-boat conformation. Comparison between these structures and the previously determined enzyme-substrate complex structure reveals that the glucose pyranose ring might be distorted immediately after nucleophilic attack by a water molecule. These structures represent the first enzyme-product complex for the GH63 family, as well as the structurally-related glycosidases, and it may provide insight into the catalytic mechanism of these enzymes.

Crystal Structure and Mutational Analysis of Isomalto-dextranase, a Member of Glycoside Hydrolase Family 27.

Arthrobacter globiformis T6 isomalto-dextranase (AgIMD) is an enzyme that liberates isomaltose from the non-reducing end of a polymer of glucose, dextran. AgIMD is classified as a member of the glycoside hydrolase family (GH) 27, which comprises mainly α-galactosidases and α-N-acetylgalactosaminidases, whereas AgIMD does not show α-galactosidase or α-N-acetylgalactosaminidase activities. Here, we determined the crystal structure of AgIMD. AgIMD consists of the following three domains: A, C, and D. Domains A and C are identified as a (ß/α)8-barrel catalytic domain and an antiparallel ß-structure, respectively, both of which are commonly found in GH27 enzymes. However, domain A of AgIMD has subdomain B, loop-1, and loop-2, all of which are not found in GH27 human α-galactosidase. AgIMD in a complex with trisaccharide panose shows that Asp-207, a residue in loop-1, is involved in subsite +1. Kinetic parameters of the wild-type and mutant enzymes for the small synthetic saccharide p-nitrophenyl α-isomaltoside and the polysaccharide dextran were compared, showing that Asp-207 is important for the catalysis of dextran. Domain D is classified as carbohydrate-binding module (CBM) 35, and an isomaltose molecule is seen in this domain in the AgIMD-isomaltose complex. Domain D is highly homologous to CBM35 domains found in GH31 and GH66 enzymes. The results here indicate that some features found in GH13, -31, and -66 enzymes, such as subdomain B, residues at the subsite +1, and the CBM35 domain, are also observed in the GH27 enzyme AgIMD and thus provide insights into the evolutionary relationships among GH13, -27, -31, -36, and -66 enzymes.

A glycoside hydrolase family 31 dextranase with high transglucosylation activity from Flavobacterium johnsoniae.

Glycoside hydrolase family (GH) 31 enzymes exhibit various substrate specificities, although the majority of members are α-glucosidases. Here, we constructed a heterologous expression system of a GH31 enzyme, Fjoh_4430, from Flavobacterium johnsoniae NBRC 14942, using Escherichia coli, and characterized its enzymatic properties. The enzyme hydrolyzed dextran and pullulan to produce isomaltooligosaccharides and isopanose, respectively. When isomaltose was used as a substrate, the enzyme catalyzed disproportionation to form isomaltooligosaccharides. The enzyme also acted, albeit inefficiently, on p-nitrophenyl α-D-glucopyranoside, and p-nitrophenyl α-isomaltoside was the main product of the reaction. In contrast, Fjoh_4430 did not act on trehalose, kojibiose, nigerose, maltose, maltotriose, or soluble starch. The optimal pH and temperature were pH 6.0 and 60 °C, respectively. Our results indicate that Fjoh_4430 is a novel GH31 dextranase with high transglucosylation activity.

Structural and biochemical characterization of novel bacterial α-galactosidases belonging to glycoside hydrolase family 31.

Glycoside hydrolase family 31 (GH31) proteins have been reportedly identified as exo-α-glycosidases with activity for α-glucosides and α-xylosides. We focused on a GH31 subfamily, which contains proteins with low sequence identity (<24%) to the previously reported GH31 glycosidases and characterized two enzymes from Pedobacter heparinus and Pedobacter saltans. The enzymes unexpectedly exhibited α-galactosidase activity, but were not active on α-glucosides and α-xylosides. The crystal structures of one of the enzymes, PsGal31A, in unliganded form and in complexes with D-galactose or L-fucose and the catalytic nucleophile mutant in unliganded form and in complex with p-nitrophenyl-α-D-galactopyranoside, were determined at 1.85-2.30 Å (1 Å=0.1 nm) resolution. The overall structure of PsGal31A contains four domains and the catalytic domain adopts a (ß/α)8-barrel fold that resembles the structures of other GH31 enzymes. Two catalytic aspartic acid residues are structurally conserved in the enzymes, whereas most residues forming the active site differ from those of GH31 α-glucosidases and α-xylosidases. PsGal31A forms a dimer via a unique loop that is not conserved in other reported GH31 enzymes; this loop is involved in its aglycone specificity and in binding L-fucose. Considering potential genes for α-L-fucosidases and carbohydrate-related proteins within the vicinity of Pedobacter Gal31, the identified Gal31 enzymes are likely to function in a novel sugar degradation system. This is the first report of α-galactosidases which belong to GH31 family.

Crystal structure and substrate-binding mode of GH63 mannosylglycerate hydrolase from Thermus thermophilus HB8.

Glycoside hydrolase family 63 (GH63) proteins are found in eukaryotes such as processing α-glucosidase I and also many bacteria and archaea. Recent studies have identified two bacterial and one plant GH63 mannosylglycerate hydrolases that act on both glucosylglycerate and mannosylglycerate, which are compatible solutes found in many thermophilic prokaryotes and some plants. Here we report the 1.67-Å crystal structure of one of these GH63 mannosylglycerate hydrolases, Tt8MGH from Thermus thermophilus HB8, which is 99% homologous to mannosylglycerate hydrolase from T. thermophilus HB27. Tt8MGH consists of a single (α/α)6-barrel catalytic domain with two additional helices and two long loops which form a homotrimer. The structures of this protein in complexes with glucose or glycerate were also determined at 1.77- or 2.10-Å resolution, respectively. A comparison of these structures revealed that the conformations of three flexible loops were largely different from each other. The conformational changes may be induced by ligand binding and serve to form finger-like structures for holding substrates. These findings represent the first-ever proposed substrate recognition mechanism for GH63 mannosylglycerate hydrolase.

Crystal structure of the catalytic domain of a GH16 ß-agarase from a deep-sea bacterium, Microbulbifer thermotolerans JAMB-A94.

A deep-sea bacterium, Microbulbifer thermotolerans JAMB-A94, has a ß-agarase (MtAgaA) belonging to the glycoside hydrolase family (GH) 16. The optimal temperature of this bacterium for growth is 43-49 °C, and MtAgaA is stable at 60 °C, which is one of the most thermostable enzymes among GH16 ß-agarases. Here, we determined the catalytic domain structure of MtAgaA. MtAgaA consists of a ß-jelly roll fold, as observed in other GH16 enzymes. The structure of MtAgaA was most similar to two ß-agarases from Zobellia galactanivorans, ZgAgaA, and ZgAgaB. Although the catalytic cleft structure of MtAgaA was similar to ZgAgaA and ZgAgaB, residues at subsite -4 of MtAgaA were not conserved between them. Also, an α-helix, designated as α4', was uniquely located near the catalytic cleft of MtAgaA. A comparison of the structures of the three enzymes suggested that multiple factors, including increased numbers of arginine and proline residues, could contribute to the thermostability of MtAgaA.