Extracellular oil production by Rhodotorula paludigena BS15 for biorefinery without complex downstream processes.

To realize biomass refinery without complex downstream processes, we extensively screened for microbial strains that efficiently produce extracellular oil from sugars. Rhodotorula paludigena (formerly Rhodosporidium paludigenum) BS15 was found to efficiently produce polyol esters of fatty acids (PEFAs), which mainly comprised of 3-acetoxypalmitic acid and partially acetylated mannitol/arabinitol. To evaluate the performance of this strain, fed-batch fermentation was demonstrated on a flask scale, and 110 g/L PEFA and 103 g/L dry cells were produced in 12 days. To the best of our knowledge, the strain BS15 exhibited the highest PEFA titer (g/L) ever to be reported so far. Because the PEFA precipitated at the bottom of the culture broth, it could be easily recovered by simply discarding the upper phase. Various carbon sources can be utilized for cell growth and/or PEFA production, which signifies the potential for converting diverse biomass sources. Two different types of next-generation sequencers, Illumina HiSeq and Oxford Nanopore PromethION, were used to analyze the whole-genome sequence of the strain BS15. The integrative data analysis generated a high-quality and reliable reference genome for PEFA-producing R. paludigena. The 22.5-M base genome sequence and the estimated genes were registered in Genbank (accession numbers BQKY01000001-BQKY01000019). KEY POINTS: • R. paludigena BS15 was isolated after an extensive screening of extracellular oil producers from natural sources. • Fed-batch fermentation of R. paludigena BS15 yielded 110 g/L of PEFA, which is the highest titer ever reported to date. • Combined analysis using Illumina and Oxford Nanopore sequencers produced the near-complete genome sequence.

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

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on ß-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 ß-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2':2,1'-ß-D-Frup (diheterolevulosan II) and ß-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2':2,1'-ß-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 ß-D-Fruf and ß-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).

Diversification of a Fucosyllactose Transporter within the Genus Bifidobacterium.

Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose [FL]), are associated with the formation of a Bifidobacterium-rich gut microbial community. Phylogenetic analysis revealed that FL transporters have diversified into two clades harboring four clusters within the Bifidobacterium genus, but the underpinning functional diversity associated with this divergence remains underexplored. In this study, we examined the HMO consumption phenotypes of two bifidobacterial species, Bifidobacterium catenulatum subsp. kashiwanohense and Bifidobacterium pseudocatenulatum, both of which possess FL-binding proteins that belong to phylogenetic clusters with unknown specificities. Growth assays, heterologous gene expression experiments, and HMO consumption analyses showed that the FL transporter type from B. catenulatum subsp. kashiwanohense JCM 15439T conferred a novel HMO uptake pattern that includes complex fucosylated HMOs (lacto-N-fucopentaose II and lacto-N-difucohexaose I/II). Further genomic landscape analyses of FL transporter-positive bifidobacterial strains revealed that the H-antigen- or Lewis antigen-specific fucosidase gene(s) and FL transporter specificities were largely aligned. These results suggest that bifidobacteria have acquired FL transporters along with the corresponding gene sets necessary to utilize the imported HMOs. Our results provide insight into the species- and strain-dependent adaptation strategies of bifidobacteria in HMO-rich environments. IMPORTANCE The gut of breastfed infants is generally dominated by health-promoting bifidobacteria. Human milk oligosaccharides (HMOs) from breast milk selectively promote the growth of specific taxa such as bifidobacteria, thus forming an HMO-mediated host-microbe symbiosis. While the coevolution of humans and bifidobacteria has been proposed, the underpinning adaptive strategies employed by bifidobacteria require further research. Here, we analyzed the divergence of the critical fucosyllactose (FL) HMO transporter within Bifidobacterium. We have shown that the diversification of the solute-binding proteins of the FL transporter led to uptake specificities of fucosylated sugars ranging from simple trisaccharides to complex hexasaccharides. This transporter and the congruent acquisition of the necessary intracellular enzymes allow bifidobacteria to consume different types of HMOs in a predictable and strain-dependent manner. These findings explain the adaptation and proliferation of bifidobacteria in the competitive and HMO-rich infant gut environment and enable accurate specificity annotation of transporters from metagenomic data.

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

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 lgdh gene from the bacterium Pseudarthrobacter phenanthrenivorans 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 Km and higher kcat 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 scyllo-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.

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

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 ß-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 (BlArap42B) from a probiotic Bifidobacterium species cannot hydrolyze ß-galactosides. However, BlArap42B effectively hydrolyzed paeonolide and ginsenoside Rb2, plant glycosides containing an aromatic aglycone conjugated to α-l-arabinopyranosyl-(1,6)-ß-d-glucopyranoside. Paeonolide, a natural glycoside from the roots of the plant genus Paeonia, is not hydrolyzed by classical GH42 ß-galactosidases. X-ray crystallography revealed a unique Trp345-X12-Trp358 sequence motif at the BlArap42B active site, as compared with a Phe-X12-His motif in classical GH42 ß-galactosidases. This analysis also indicated that the C6 position of galactose is blocked by the aromatic side chains, hence allowing accommodation only of Arap lacking this carbon. Automated docking of paeonolide revealed that it can fit into the BlArap42B active site. The Glcp moiety of paeonolide stacks onto the aromatic ring of the Trp252 at subsite +1 and C4-OH is hydrogen bonded with Asp249 Moreover, the aglycone stacks against Phe421 from the neighboring monomer in the BlArap42B 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.

The crystal structure of an inverting glycoside hydrolase family 9 exo-ß-D-glucosaminidase and the design of glycosynthase.

Exo-ß-D-glucosaminidase (EC 3.2.1.165) from Photobacterium profundum (PpGlcNase) is an inverting GH (glycoside hydrolase) belonging to family 9. We have determined the three-dimensional structure of PpGlcNase to describe the first structure-function relationship of an exo-type GH9 glycosidase. PpGlcNase has a narrow and straight active-site pocket, in contrast with the long glycan-binding cleft of a GH9 endoglucanase. This is because PpGlcNase has a long loop, which blocks the position corresponding to subsites -4 to -2 of the endoglucanase. The pocket shape of PpGlcNase explains its substrate preference for a ß1,4-linkage at the non-reducing terminus. Asp(139), Asp(143) and Glu(555) in the active site were located near the ß-O1 hydroxy group of GlcN (D-glucosamine), with Asp(139) and Asp(143) holding a nucleophilic water molecule for hydrolysis. The D139A, D143A and E555A mutants significantly decreased hydrolytic activity, indicating their essential role. Of these mutants, D139A exclusively exhibited glycosynthase activity using α-GlcN-F (α-D-glucosaminyl fluoride) and GlcN as substrates, to produce (GlcN)2. Using saturation mutagenesis at Asp(139), we obtained D139E as the best glycosynthase. Compared with the wild-type, the hydrolytic activity of D139E was significantly suppressed (<0.1%), and the F(-)-release activity also decreased (<3%). Therefore the glycosynthase activity of D139E was lower than that of glycosynthases created previously from other inverting GHs. Mutation at the nucleophilic water holder is a general strategy for creating an effective glycosynthase from inverting GHs. However, for GH9, where two acidic residues seem to share the catalytic base role, mutation of Asp(139) might inevitably reduce F(-)-release activity.

Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase, Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes.

The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes.

Open-close structural change upon ligand binding and two magnesium ions required for the catalysis of N-acetylhexosamine 1-kinase.

Infant gut-associated bifidobacteria possess a metabolic pathway to utilize lacto-N-biose (Gal-ß1,3-GlcNAc) and galacto-N-biose (Gal-ß1,3-GalNAc) from human milk and glycoconjugates specifically. In this pathway, N-acetylhexosamine 1-kinase (NahK) catalyzes the phosphorylation of GlcNAc or GalNAc at the anomeric C1 position with ATP. Crystal structures of NahK have only been determined in the closed state. In this study, we determined open state structures of NahK in three different forms (apo, ADP complex, and ATP complex). A comparison of the open and closed state structures revealed an induced fit structural change defined by two rigid domains. ATP binds to the small N-terminal domain, and binding of the N-acetylhexosamine substrate to the large C-terminal domain induces a closing conformational change with a rotation angle of 16°. In the nucleotide binding site, two magnesium ions bridging the α-γ and ß-γ phosphates were identified. A mutational analysis indicated that a residue coordinating both of the two magnesium ions (Asp228) is essential for catalysis. The involvement of two magnesium ions in the catalytic machinery is structurally similar to the catalytic structures of protein kinases and aminoglycoside phosphotransferases, but distinct from the structures of other anomeric kinases or sugar 6-kinases. These findings help to elucidate the possible evolutionary adaptation of substrate specificities and induced fit mechanism.

Introduction of H-antigens into oligosaccharides and sugar chains of glycoproteins using highly efficient 1,2-α-l-fucosynthase.

Fucα1-2 Gal linkages, or H-antigens, constitute histo-blood group antigens and are involved in various physiological processes. In addition, recent studies have shown that the H-antigen-containing glycans play an important role, not only in establishing harmonious relationship between gut microbes and the host, but also in preventing gut dysbiosis-related diseases. Therefore, development of an efficient method for introducing Fuc residue at Gal residue at the nonreducing end of glycans via α-(1→2) linkage is desired for research as well as medicinal purposes. In this study, we succeeded in derivatizing inverting 1,2-α-l-fucosidase (AfcA) into a highly efficient 1,2-α-l-fucosynthase. The synthase specifically synthesized H type 1-, type 2-, type 3- and type 4-chain-containing oligosaccharides with yields of 57-75% based on acceptor depletion. The synthase was also able to specifically introduce Fuc residues into Lewis a/x antigens to produce Lewis b/y antigens, with yields of 43% and 62%, respectively. In addition, the enzyme efficiently introduced H-antigens into sugar chains of porcine gastric mucins, as revealed by lectin blotting and mass spectroscopy analysis of the sugars. Detailed acceptor specificity analysis using various monosaccharides and oligosaccharides unraveled unique substrate recognition feature of this synthase at the subsite (+1), which can be explained by our previous X-ray crystallographic study of AfcA. These results show that the synthase developed in this study could serve as an alternative to other H-antigen synthesis methods involving α-1,2-fucosyltransferases and retaining α-fucosidase.

Structural basis for reversible phosphorolysis and hydrolysis reactions of 2-O-α-glucosylglycerol phosphorylase.

2-O-α-Glucosylglycerol phosphorylase (GGP) from Bacillus selenitireducens catalyzes both the reversible phosphorolysis of 2-O-α-glucosylglycerol (GG) and the hydrolysis of ß-d-glucose 1-phosphate (ßGlc1P). GGP belongs to the glycoside hydrolase (GH) family 65 and can efficiently and specifically produce GG. However, its structural basis has remained unclear. In this study, the crystal structures of GGP complexed with glucose and the glucose analog isofagomine and glycerol were determined. Subsite -1 of GGP is similar to those of other GH65 enzymes, maltose phosphorylase and kojibiose phosphorylase, whereas subsite +1 is largely different and is well designed for GG recognition. An automated docking analysis was performed to complement these crystal structures, ßGlc1P being docked at an appropriate position. To investigate the importance of residues at subsite +1 in the bifunctionality of GGP, we constructed mutants at these residues. Y327F and K587A did not show detectable activities for either reverse phosphorolysis or ßGlc1P hydrolysis. Y572F also showed significantly reduced activities for both of these reactions. In contrast, W381F showed significantly reduced reverse phosphorolytic activity but retained ßGlc1P hydrolysis. The mode of substrate recognition and the reaction mechanisms of GGP were proposed based on these analyses. Specifically, an extensive hydrogen bond network formed by Tyr-327, Tyr-572, Lys-587, and water molecules contributes to fixing the acceptor molecule in both reverse phosphorolysis (glycerol) and ßGlc1P hydrolysis (water) for a glycosyl transfer reaction. This study will contribute to the development of a large scale production system of GG by facilitating the rational engineering of GGP.

A ß1-6/ß1-3 galactosidase from Bifidobacterium animalis subsp. lactis†Bl-04 gives insight into sub-specificities of ß-galactoside catabolism within Bifidobacterium.

The Bifidobacterium genus harbours several health promoting members of the gut microbiota. Bifidobacteria display metabolic specialization by preferentially utilizing dietary or host-derived ß-galactosides. This study investigates the biochemistry and structure of a glycoside hydrolase family 42 (GH42) ß-galactosidase from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 (BlGal42A). BlGal42A displays a preference for undecorated ß1-6 and ß1-3 linked galactosides and populates a phylogenetic cluster with close bifidobacterial homologues implicated in the utilization of N-acetyl substituted ß1-3 galactosides from human milk and mucin. A long loop containing an invariant tryptophan in GH42, proposed to bind substrate at subsite + 1, is identified here as specificity signature within this clade of bifidobacterial enzymes. Galactose binding at the subsite - 1 of the active site induced conformational changes resulting in an extra polar interaction and the ordering of a flexible loop that narrows the active site. The amino acid sequence of this loop provides an additional specificity signature within this GH42 clade. The phylogenetic relatedness of enzymes targeting ß1-6 and ß1-3 galactosides likely reflects structural differences between these substrates and ß1-4 galactosides, containing an axial galactosidic bond. These data advance our molecular understanding of the evolution of sub-specificities that support metabolic specialization in the gut niche.

Diversity of phosphorylases in glycoside hydrolase families.

Phosphorylases are useful catalysts for the practical preparation of various sugars. The number of known specificities was 13 in 2002 and is now 30. The drastic increase in available genome sequences has facilitated the discovery of novel activities. Most of these novel phosphorylase activities have been identified through the investigations of glycoside hydrolase families containing known phosphorylases. Here, the diversity of phosphorylases in each family is described in detail.

Functional reassignment of Cellvibrio vulgaris EpiA to cellobiose 2-epimerase and an evaluation of the biochemical functions of the 4-O-ß-D-mannosyl-D-glucose phosphorylase-like protein, UnkA.

The aerobic soil bacterium Cellvibrio vulgaris has a ß-mannan-degradation gene cluster, including unkA, epiA, man5A, and aga27A. Among these genes, epiA has been assigned to encode an epimerase for converting D-mannose to D-glucose, even though the amino acid sequence of EpiA is similar to that of cellobiose 2-epimerases (CEs). UnkA, whose function currently remains unknown, shows a high sequence identity to 4-O-ß-D-mannosyl-D-glucose phosphorylase. In this study, we have investigated CE activity of EpiA and the general characteristics of UnkA using recombinant proteins from Escherichia coli. Recombinant EpiA catalyzed the epimerization of the 2-OH group of sugar residue at the reducing end of cellobiose, lactose, and ß-(1→4)-mannobiose in a similar manner to other CEs. Furthermore, the reaction efficiency of EpiA for ß-(1→4)-mannobiose was 5.5 × 10(4)-fold higher than it was for D-mannose. Recombinant UnkA phosphorolyzed ß-D-mannosyl-(1→4)-D-glucose and specifically utilized D-glucose as an acceptor in the reverse reaction, which indicated that UnkA is a typical 4-O-ß-D-mannosyl-D-glucose phosphorylase.

Discovery of ß-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase involved in the metabolism of N-glycans.

A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, ß-N-acetylhexosaminidase, exo-α-sialidase, and endo-ß-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of ß-1,4-D-mannosyl-N-acetyl-D-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where ß-1,4-D-mannosyl-N-acetyl-D-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-D-mannose 1-phosphate and N-acetyl-D-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where ß-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the D-mannose residue of ß-1,4-D-mannosyl-N-acetyl-D-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-ß-D-mannopyranosyl-N-acetyl-D-glucosamine:phosphate α-D-mannosyltransferase as the systematic name and ß-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase as the short name for BT1033.

Lacto-N-biosidase encoded by a novel gene of Bifidobacterium longum subspecies longum shows unique substrate specificity and requires a designated chaperone for its active expression.

Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galß1-3GlcNAcß1-3Galß1-4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galß1-3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase(+) strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcß1-3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1-2Galß1-3GlcNAcß1-3Galß1-4Glc), and sialyllacto-N-tetraose a (Neu5Acα2-3Galß1-3GlcNAcß1-3Galß1-4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-ß-lacto-N-bioside I (Galß1-3GlcNAcß-pNP) and GalNAcß1-3GlcNAcß-pNP. GalNAcß1-3GlcNAcß linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca(2+) and Mg(2+)) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.

Distinct substrate specificities of three glycoside hydrolase family 42 ß-galactosidases from Bifidobacterium longum subsp. infantis ATCC 15697.

Glycoside hydrolase family 42 (GH42) includes ß-galactosidases catalyzing the release of galactose (Gal) from the non-reducing end of different ß-d-galactosides. Health-promoting probiotic bifidobacteria, which are important members of the human gastrointestinal tract microbiota, produce GH42 enzymes enabling utilization of ß-galactosides exerting prebiotic effects. However, insight into the specificity of individual GH42 enzymes with respect to substrate monosaccharide composition, glycosidic linkage and degree of polymerization is lagging. Kinetic analysis of natural and synthetic substrates resembling various milk and plant galactooligosaccharides distinguishes the three GH42 members, Bga42A, Bga42B and Bga42C, encoded by the probiotic B. longum subsp. infantis ATCC 15697 and revealed the glycosyl residue at subsite +1 and its linkage to the terminal Gal at subsite -1 to be key specificity determinants. Bga42A thus prefers the ß1-3-galactosidic linkage from human milk and other ß1-3- and ß1-6-galactosides with glucose or Gal situated at subsite +1. In contrast, Bga42B very efficiently hydrolyses 4-galactosyllactose (Galß1-4Galß1-4Glc) as well as 4-galactobiose (Galß1-4Gal) and 4-galactotriose (Galß1-4Galß1-4Gal). The specificity of Bga42C resembles that of Bga42B, but the activity was one order of magnitude lower. Based on enzyme kinetics, gene organization and phylogenetic analyses, Bga42C is proposed to act in the metabolism of arabinogalactan-derived oligosaccharides. The distinct kinetic signatures of the three GH42 enzymes correlate to unique sequence motifs denoting specific clades in a GH42 phylogenetic tree providing novel insight into GH42 subspecificities. Overall, the data illustrate the metabolic adaptation of bifidobacteria to the ß-galactoside-rich gut niche and emphasize the importance and diversity of ß-galactoside metabolism in probiotic bifidobacteria.

Characterization of a thermophilic 4-O-ß-D-mannosyl-D-glucose phosphorylase from Rhodothermus marinus.

4-O-ß-D-Mannosyl-D-glucose phosphorylase (MGP), found in anaerobes, converts 4-O-ß-D-mannosyl-D-glucose (Man-Glc) to α-D-mannosyl phosphate and D-glucose. It participates in mannan metabolism with cellobiose 2-epimerase (CE), which converts ß-1,4-mannobiose to Man-Glc. A putative MGP gene is present in the genome of the thermophilic aerobe Rhodothermus marinus (Rm) upstream of the gene encoding CE. Konjac glucomannan enhanced production by R. marinus of MGP, CE, and extracellular mannan endo-1,4-ß-mannosidase. Recombinant RmMGP catalyzed the phosphorolysis of Man-Glc through a sequential bi-bi mechanism involving ternary complex formation. Its molecular masses were 45 and 222 kDa under denaturing and nondenaturing conditions, respectively. Its pH and temperature optima were 6.5 and 75 °C, and it was stable between pH 5.5-8.3 and below 80 °C. In the reverse reaction, RmMGP had higher acceptor preferences for 6-deoxy-D-glucose and D-xylose than R. albus NE1 MGP. In contrast to R. albus NE1 MGP, RmMGP utilized methyl ß-D-glucoside and 1,5-anhydro-D-glucitol as acceptor substrates.

Molecular Basis of Absorption at 340 nm of 3-Ketoglucosides under Alkaline Conditions.

Transient absorption at 340 nm under alkaline conditions has long been used to detect the presence of 3-keto-O-glycosides without understanding the molecular basis of the absorbance. The time course of A340 nm for the alkaline treatment of 3-ketolevoglucosan, an intramolecular 3-keto-O-glycoside, was investigated to identify the three products generated through alkaline treatment. By comparing the spectra of these compounds under neutral and alkaline conditions, we identified 1,5-anhydro-D-erythro-hex-1-en-3-ulose (2-hydroxy-3-keto-D-glucal) as being the compound responsible for the absorption.

α-N-acetylgalactosaminidase from infant-associated bifidobacteria belonging to novel glycoside hydrolase family 129 is implicated in alternative mucin degradation pathway.

Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo-α-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Galß1-3GalNAcα1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core-3-type O-glycans (GlcNAcß1-3GalNAcα1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel α-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAcα1-Ser/Thr). Sialyl and galactosyl core-3 (Galß1-3/4GlcNAcß1-3(Neu5Acα2-6)GalNAcα1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as α-sialidase (SiaBb2), lacto-N-biosidase (LnbB), ß-galactosidase (BbgIII), and ß-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129.