scholarly journals Trehalose Degradation by Cellvibrio japonicus Exhibits No Functional Redundancy and Is Solely Dependent on the Tre37A Enzyme

2020 ◽  
Vol 86 (22) ◽  
Author(s):  
Cecelia A. Garcia ◽  
Jackson A. Narrett ◽  
Jeffrey G. Gardner
Keyword(s):  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Glycoside Hydrolase Family ◽  
Gut Health ◽  
Phosphotransferase System ◽  
Carbohydrate Active Enzymes ◽  
Human Gut ◽  
Content Type ◽  
Degrading Bacteria ◽  
Cellvibrio Japonicus

ABSTRACT The α-diglucoside trehalose has historically been known as a component of the bacterial stress response, though it more recently has been studied for its relevance in human gut health and biotechnology development. The utilization of trehalose as a nutrient source by bacteria relies on carbohydrate-active enzymes, specifically those of the glycoside hydrolase family 37 (GH37), to degrade the disaccharide into substituent glucose moieties for entry into metabolism. Environmental bacteria using oligosaccharides for nutrients often possess multiple carbohydrate-active enzymes predicted to have the same biochemical activity and therefore are thought to be functionally redundant. In this study, we characterized trehalose degradation by the biotechnologically important saprophytic bacterium Cellvibrio japonicus. This bacterium possesses two predicted α-α-trehalase genes, tre37A and tre37B, and our investigation using mutational analysis found that only the former is essential for trehalose utilization by C. japonicus. Heterologous expression experiments found that only the expression of the C. japonicus tre37A gene in an Escherichia coli treA mutant strain allowed for full utilization of trehalose. Biochemical characterization of C. japonicus GH37 activity determined that the tre37A gene product is solely responsible for cleaving trehalose and is an acidic α-α-trehalase. Bioinformatic and mutational analyses indicate that Tre37A directly cleaves trehalose to glucose in the periplasm, as C. japonicus does not possess a phosphotransferase system. This study facilitates the development of a comprehensive metabolic model for α-linked disaccharides in C. japonicus and more broadly expands our understanding of the strategies that saprophytic bacteria employ to capture diverse carbohydrates from the environment. IMPORTANCE The metabolism of trehalose is becoming increasingly important due to the inclusion of this α-diglucoside in a number of foods and its prevalence in the environment. Bacteria able to utilize trehalose in the human gut possess a competitive advantage, as do saprophytic microbes in terrestrial environments. While the biochemical mechanism of trehalose degradation is well understood, what is less clear is how bacteria acquire this metabolite from the environment. The significance of this report is that by using the model saprophyte Cellvibrio japonicus, we were able to functionally characterize the two predicted trehalase enzymes that the bacterium possesses and determined that the two enzymes are not equivalent and are not functionally redundant. The results and approaches used to understand the complex physiology of α-diglucoside metabolism from this study can be applied broadly to other polysaccharide-degrading bacteria.

scholarly journals Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Cathleen Kmezik ◽  
Daniel Krska ◽  
Scott Mazurkewich ◽  
Johan Larsbrink
Keyword(s):  
Biochemical Characterization ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Active Enzymes ◽  
Human Gut ◽  
Glucuronoyl Esterase ◽  
Cob Biomass ◽  
Polysaccharide Utilization Loci ◽  
Hydrolase Family ◽  
Discrete Functions

AbstractBacteroidetes are efficient degraders of complex carbohydrates, much thanks to their use of polysaccharide utilization loci (PULs). An integral part of PULs are highly specialized carbohydrate-active enzymes, sometimes composed of multiple linked domains with discrete functions—multicatalytic enzymes. We present the biochemical characterization of a multicatalytic enzyme from a large PUL encoded by the gut bacterium Bacteroides eggerthii. The enzyme, BeCE15A-Rex8A, has a rare and novel architecture, with an N-terminal carbohydrate esterase family 15 (CE15) domain and a C-terminal glycoside hydrolase family 8 (GH8) domain. The CE15 domain was identified as a glucuronoyl esterase (GE), though with relatively poor activity on GE model substrates, attributed to key amino acid substitutions in the active site compared to previously studied GEs. The GH8 domain was shown to be a reducing-end xylose-releasing exo-oligoxylanase (Rex), based on having activity on xylooligosaccharides but not on longer xylan chains. The full-length BeCE15A-Rex8A enzyme and the Rex domain were capable of boosting the activity of a commercially available GH11 xylanase on corn cob biomass. Our research adds to the understanding of multicatalytic enzyme architectures and showcases the potential of discovering novel and atypical carbohydrate-active enzymes from mining PULs.

scholarly journals Wood-Derived Dietary Fibers Promote Beneficial Human Gut Microbiota

mSphere ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Sabina Leanti La Rosa ◽  
Vasiliki Kachrimanidou ◽  
Fanny Buffetto ◽  
Phillip B. Pope ◽  
Nicholas A. Pudlo ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Well Being ◽  
Gut Health ◽  
Human Gut ◽  
Content Type ◽  
Human Gut Microbiota ◽  
Lignocellulosic Feedstocks ◽  
Host Health ◽  
Clostridial Cluster ◽  
Gut Microbiota Composition

The architecture of the gut bacterial ecosystem has a profound effect on the physiology and well-being of the host. Modulation of the gut microbiota and the intestinal microenvironment via administration of prebiotics represents a valuable strategy to promote host health. This work provides insights into the ability of two novel wood-derived preparations, AcGGM and AcAGX, to influence human gut microbiota composition and activity. These compounds were selectively fermented by commensal bacteria such as Bifidobacterium, Bacteroides-Prevotella, F. prausnitzii, and clostridial cluster IX spp. This promoted the microbial synthesis of acetate, propionate, and butyrate, which are beneficial to the microbial ecosystem and host colonic epithelial cells. Thus, our results demonstrate potential prebiotic properties for both AcGGM and AcAGX from lignocellulosic feedstocks. These findings represent pivotal requirements for rationally designing intervention strategies based on the dietary supplementation of AcGGM and AcAGX to improve or restore gut health.

scholarly journals Cleavage of Rubber by the Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30: Molecular Insights

2016 ◽  
Vol 82 (22) ◽  
pp. 6593-6602 ◽  
Author(s):  
Wolf Röther ◽  
Stefanie Austen ◽  
Jakob Birke ◽  
Dieter Jendrossek
Keyword(s):  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Axial Position ◽  
Amino Acid Residues ◽  
Content Type ◽  
Research Activities ◽  
Domain Of Unknown Function ◽  
Arginine Residues ◽  
Aldehyde Groups ◽  
Key Residues

ABSTRACTGram-positive rubber degraders such asStreptomycessp. strain K30 cleave rubber [poly(cis-1,4-isoprene)] to low-molecular-mass oligoisoprenoid products with terminal keto and aldehyde groups by the secretion of a latex clearing protein (Lcp) designated rubber oxygenase. LcpK30is a hemebcytochrome and has a domain of unknown function (DUF2236) that is characteristic of orthologous Lcps. Proteins with a DUF2236 domain are characterized by three highly conserved residues (R164, T168, and H198 in LcpK30). Exchange of R164 or T168 by alanine and characterization of the purified LcpK30muteins revealed that both were stable and contained a heme group (red color) but were inactive. This finding identifies both residues as key residues for the cleavage reaction. The purified H198A mutein was also inactive and stable but was colorless due to the absence of heme. We constructed and characterized alanine muteins of four additional histidine residues moderately conserved in 495 LcpK30homologous sequences (H203A, H232A, H259A, H266A). All muteins revealed wild-type properties, excluding any importance for activity and/or heme coordination. Since LcpK30has only eight histidines and the three remaining residues (H103, H184, and H296) were not conserved (<11%), H198 presumably is the only essential histidine, indicating its putative function as a heme ligand. The second axial position of the heme is likely occupied by a not yet identified molecule. Mutational analysis of three strictly conserved arginine residues (R195, R202, R328) showed that R195A and R202A muteins were colorless and instable, suggesting that these residues are important for the protein stability.IMPORTANCELarge amounts of rubber waste materials have been permanently released into the environment for more than a century, yet accumulation of rubber particles released, e.g., by abrasion of tires along highways has not been observed. This is indicative of the ubiquitous presence and activity of rubber-degrading microorganisms. Despite increasing research activities on rubber biodegradation during the last 2 decades, the knowledge of the enzymatic cleavage mechanism of rubber by latex clearing protein (Lcp) still is limited. In particular, the catalytic cleavage mechanism and the amino acids of Lcp proteins (Lcps) that are involved have not yet been identified for any Lcp. In this study, we investigated the importance of 10 amino acid residues of Lcp fromStreptomycessp. K30 (LcpK30) by mutagenesis, mutein purification, and biochemical characterization. We identified several essential residues, one of which most likely represents an axial heme ligand in Lcp ofStreptomycessp. K30.

scholarly journals Biochemical and structural analyses of a bacterial endo-β-1,2-glucanase reveal a new glycoside hydrolase family

2017 ◽  
Vol 292 (18) ◽  
pp. 7487-7506 ◽  
Author(s):  
Koichi Abe ◽  
Masahiro Nakajima ◽  
Tetsuro Yamashita ◽  
Hiroki Matsunaga ◽  
Shinji Kamisuki ◽  
...  
Keyword(s):  
Active Site ◽  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Sequence Similarity ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Significant Sequence Similarity ◽  
Cell Extracts ◽  
Ligand Free ◽  
Complex Forms

β-1,2-Glucan is an extracellular cyclic or linear polysaccharide from Gram-negative bacteria, with important roles in infection and symbiosis. Despite β-1,2-glucan's importance in bacterial persistence and pathogenesis, only a few reports exist on enzymes acting on both cyclic and linear β-1,2-glucan. To this end, we purified an endo-β-1,2-glucanase to homogeneity from cell extracts of the environmental species Chitinophaga arvensicola, and an endo-β-1,2-glucanase candidate gene (Cpin_6279) was cloned from the related species Chitinophaga pinensis. The Cpin_6279 protein specifically hydrolyzed linear β-1,2-glucan with polymerization degrees of ≥5 and a cyclic counterpart, indicating that Cpin_6279 is an endo-β-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-β-1,2-Glc-β-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 β-1,2-glucanases and represent a critical advance toward elucidating structure-function relationships of GH enzymes.

scholarly journals Cross-Feeding among Probiotic Bacterial Strains on Prebiotic Inulin Involves the Extracellularexo-Inulinase ofLactobacillus paracaseiStrain W20

2018 ◽  
Vol 84 (21) ◽  
Author(s):  
Markus C. L. Boger ◽  
Alicia Lammerts van Bueren ◽  
Lubbert Dijkhuizen
Keyword(s):  
Degradation Products ◽  
Lactobacillus Paracasei ◽  
Transport Systems ◽  
Glycoside Hydrolase Family ◽  
Bacterial Strains ◽  
Human Gut ◽  
Content Type ◽  
Beneficial Effects ◽  
Probiotic Lactobacilli

ABSTRACTProbiotic gut bacteria employ specific metabolic pathways to degrade dietary carbohydrates beyond the capabilities of their human host. Here, we report how individual commercial probiotic strains degrade prebiotic (inulin type) fructans. First, a structural analysis of commercial fructose oligosaccharide-inulin samples was performed. These β-(2-1)-fructans differ in termination by either glucose (GF) or fructose (FF) residues, with a broad variation in the degrees of polymerization (DPs). The growth of individual probiotic bacteria on short-chain inulin (sc-inulin) (Frutafit CLR), a β-(2-1)-fructan (DP 2 to DP 40), was studied.Lactobacillus salivariusW57 and other bacteria grew relatively poorly on sc-inulin, with only fractions of DP 3 and DP 5 utilized, reflecting uptake via specific transport systems followed by intracellular metabolism.Lactobacillus paracaseisubsp.paracaseiW20 completely used all sc-inulin components, employing an extracellularexo-inulinase enzyme (glycoside hydrolase family GH32 [LpGH32], also found in other strains of this species); the purified enzyme converted high-DP compounds into fructose, sucrose, 1-kestose, and F2 (inulobiose). The cocultivation ofL. salivariusW57 andL. paracaseiW20 on sc-inulin resulted in cross-feeding of the former by the latter, supported by this extracellularexo-inulinase. The extent of cross-feeding depended on the type of fructan, i.e., the GF type (clearly stimulating) versus the FF type (relatively low stimulus), and on fructan chain length, since relatively low-DP β-(2-1)-fructans contain a relatively high content of GF-type molecules, thus resulting in higher concentrations of GF-type DP 2 to DP 3 degradation products. The results provide an example of howin vivocross-feeding on prebiotic β-(2-1)-fructans may occur among probiotic lactobacilli.IMPORTANCEThe human gut microbial community is associated strongly with host physiology and human diseases. This observation has prompted research on pre- and probiotics, two concepts enabling specific changes in the composition of the human gut microbiome that result in beneficial effects for the host. Here, we show how fructooligosaccharide-inulin prebiotics are fermented by commercial probiotic bacterial strains involving specific sets of enzymes and transporters. Cross-feeding strains such asLactobacillus paracaseiW20 may thus act as keystone strains in the degradation of prebiotic inulin in the human gut, and this strain–exo-inulinase combination may be used in commercialLactobacillus-inulin synbiotics.

scholarly journals Characterization of a galactosyl-binding protein module from a Cellvibrio japonicus endo-xyloglucanase defines a new family of Carbohydrate Binding Modules

2020 ◽  
pp. AEM.02634-20
Author(s):  
Mohamed A. Attia ◽  
Harry Brumer
Keyword(s):  
Binding Protein ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Substrate Affinity ◽  
Carbohydrate Binding ◽  
Carbohydrate Active Enzymes ◽  
Plant Polysaccharides ◽  
New Family ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus

Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, e.g. by increasing substrate affinity. The Gram-negative soil saprophyte Cellvibrio japonicus is valuable source for CAZyme and CBM discovery and characterization, due to its innate ability to degrade a wide array of plant polysaccharides. Bioinformatic analysis of the CJA_2959 gene product from C. japonicus revealed a modular architecture consisting of a fibronectin type III (Fn3) module, a cryptic module of unknown function (“X181”), and a Glycoside Hydrolase Family 5 subfamily 4 (GH5_4) catalytic module. We previously demonstrated that the last of these, CjGH5F, is an efficient and specific endo-xyloglucanase [Attia et al. 2018. Biotechnol. Biofuels, 11: 45]. In the present study, C-terminal fusion of superfolder green fluorescent protein in tandem with the Fn3-X181 modules enabled recombinant production and purification from Escherichia coli. Native affinity gel electrophoresis revealed binding specificity for the terminal galactose-containing plant polysaccharides galactoxyloglucan and galactomannan. Isothermal titration calorimetry further evidenced a preference for galactoxyloglucan polysaccharide over short oligosaccharides comprising the limit-digest product of CjGH5F. Thus, our results identify the X181 module as the defining member of a new CBM family, CBM88. In addition to directly revealing the function of this CBM in the context of xyloglucan metabolism by C. japonicus, this study will guide future bioinformatic and functional analyses across microbial (meta)genomes.Importance This study reveals Carbohydrate Binding Module Family 88 (CBM88) as a new family of galactose-binding protein modules, which are found in series with diverse microbial glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. The definition of CBM88 in the Carbohydrate-Active Enzymes classification (http://www.cazy.org/CBM88.html) will significantly enable future microbial (meta)genome analysis and functional studies.

scholarly journals The Phylogenomic Diversity of Herbivore-AssociatedFibrobacterspp. Is Correlated to Lignocellulose-Degrading Potential

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Anthony P. Neumann ◽  
Garret Suen
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Phylogenetic Diversity ◽  
Plant Cell Wall ◽  
Carbohydrate Active Enzymes ◽  
Content Type ◽  
Carbohydrate Active Enzyme ◽  
Functional Differences ◽  
Degrading Bacteria ◽  
Genes Encoding

ABSTRACTMembers of the genusFibrobacterare cellulose-degrading bacteria and common constituents of the gastrointestinal microbiota of herbivores. Although considerable phylogenetic diversity is observed among members of this group, few functional differences explaining the distinct ecological distributions of specific phylotypes have been described. In this study, we sequenced and performed a comparative analysis of whole genomes from 38 novelFibrobacterstrains against the type strains for the two formally describedFibrobacterspeciesF. succinogenesstrain S85 andF. intestinalisstrain NR9. Significant differences in the number of genes encoding carbohydrate-active enzyme families involved in plant cell wall polysaccharide degradation were observed amongFibrobacterphylotypes.F. succinogenesgenomes were consistently enriched in genes encoding carbohydrate-active enzymes compared to those ofF. intestinalisstrains. Moreover, genomes ofF. succinogenesphylotypes that are dominant in the rumen had significantly more genes annotated to major families involved in hemicellulose degradation (e.g., CE6, GH10, and GH43) than did the genomes ofF. succinogenesphylotypes typically observed in the lower gut of large hindgut-fermenting herbivores such as horses. Genes encoding a putative urease were also identified in 12 of theFibrobactergenomes, which were primarily isolated from hindgut-fermenting hosts. Screening for growth on urea as the sole source of nitrogen provided strong evidence that the urease was active in these strains. These results represent the strongest evidence reported to date for specific functional differences contributing to the ecology ofFibrobacterspp. in the herbivore gut.IMPORTANCEThe herbivore gut microbiome is incredibly diverse, and a functional understanding of this diversity is needed to more reliably manipulate this community for specific gain, such as increased production in ruminant livestock. Microbial degraders of plant cell wall polysaccharides in the herbivore gut, particularlyFibrobacterspp., are of fundamental importance to their hosts for digestion of a diet consisting primarily of recalcitrant plant fibers. Considerable phylogenetic diversity exists among members of the genusFibrobacter, but much of this diversity remains cryptic. Here, we used comparative genomics, applied to a diverse collection of recently isolatedFibrobacterstrains, to identify a robust association between carbohydrate-active enzyme gene content and theFibrobacterphylogeny. Our results provide the strongest evidence reported to date for functional differences amongFibrobacterphylotypes associated with either the rumen or the hindgut and emphasize the general significance of carbohydrate-active enzymes in the evolution of fiber-degrading bacteria.

scholarly journals Functional Analysis of Family GH36 α-Galactosidases from Ruminococcus gnavus E1: Insights into the Metabolism of a Plant Oligosaccharide by a Human Gut Symbiont

2012 ◽  
Vol 78 (21) ◽  
pp. 7720-7732 ◽  
Author(s):  
M. Cervera-Tison ◽  
L. E. Tailford ◽  
C. Fuell ◽  
L. Bruel ◽  
G. Sulzenbacher ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Glycoside Hydrolase ◽  
Analytical Ultracentrifugation ◽  
Common Species ◽  
Phosphotransferase System ◽  
Human Gut ◽  
Content Type ◽  
Encoding Gene ◽  
Strict Specificity

ABSTRACTRuminococcus gnavusbelongs to the 57 most common species present in 90% of individuals. Previously, we identified an α-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 fromR. gnavusE1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14–21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed itaga2. Althoughaga1showed a very simple genetic organization,aga2is part of an operon of unique structure, including genes putatively encoding a regulator, a GH13, two phosphotransferase system (PTS) sequences, and a GH32, probably involved in extracellular and intracellular sucrose assimilation. The 727-amino-acid (aa) deduced Aga2 protein shares approximately 45% identity with Aga1. Both Aga1 and Aga2 expressed inEscherichia colishowed strict specificity for α-linked galactose. Both enzymes were active on natural substrates such as melibiose, raffinose, and stachyose. Aga1 and Aga2 occurred as homotetramers in solution, as shown by analytical ultracentrifugation. Modeling of Aga1 and Aga2 identified key amino acids which may be involved in substrate specificity and stabilization of the α-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with α-(1,6) regioselectivity, leading to the formation of product structures up to [Hex]12and [Hex]8, respectively. We suggest that Aga1 and Aga2 play essential roles in the metabolism of dietary oligosaccharides and could be used for the design of galacto-oligosaccharide (GOS) prebiotics, known to selectively modulate the beneficial gut microbiota.

scholarly journals An Extracellular Cell-Attached Pullulanase Confers Branched α-Glucan Utilization in Human Gut Lactobacillus acidophilus

2017 ◽  
Vol 83 (12) ◽  
Author(s):  
Marie S. Møller ◽  
Yong Jun Goh ◽  
Kasper Bøwig Rasmussen ◽  
Wojciech Cypryk ◽  
Hasan Ufuk Celebioglu ◽  
...  
Keyword(s):  
Lactobacillus Acidophilus ◽  
Modular Organization ◽  
Glycoside Hydrolase Family ◽  
Substrate Affinity ◽  
Starch Metabolism ◽  
Growth Data ◽  
Human Gut ◽  
Content Type ◽  
Dietary Starch

ABSTRACT Of the few predicted extracellular glycan-active enzymes, glycoside hydrolase family 13 subfamily 14 (GH13_14) pullulanases are the most common in human gut lactobacilli. These enzymes share a unique modular organization, not observed in other bacteria, featuring a catalytic module, two starch binding modules, a domain of unknown function, and a C-terminal surface layer association protein (SLAP) domain. Here, we explore the specificity of a representative of this group of pullulanases, Lactobacillus acidophilus Pul13_14 (LaPul13_14), and its role in branched α-glucan metabolism in the well-characterized Lactobacillus acidophilus NCFM, which is widely used as a probiotic. Growth experiments with L. acidophilus NCFM on starch-derived branched substrates revealed a preference for α-glucans with short branches of about two to three glucosyl moieties over amylopectin with longer branches. Cell-attached debranching activity was measurable in the presence of α-glucans but was repressed by glucose. The debranching activity is conferred exclusively by LaPul13_14 and is abolished in a mutant strain lacking a functional LaPul13_14 gene. Hydrolysis kinetics of recombinant LaPul13_14 confirmed the preference for short-branched α-glucan oligomers consistent with the growth data. Curiously, this enzyme displayed the highest catalytic efficiency and the lowest Km reported for a pullulanase. Inhibition kinetics revealed mixed inhibition by β-cyclodextrin, suggesting the presence of additional glucan binding sites besides the active site of the enzyme, which may contribute to the unprecedented substrate affinity. The enzyme also displays high thermostability and higher activity in the acidic pH range, reflecting adaptation to the physiologically challenging conditions in the human gut. IMPORTANCE Starch is one of the most abundant glycans in the human diet. Branched α-1,6-glucans in dietary starch and glycogen are nondegradable by human enzymes and constitute a metabolic resource for the gut microbiota. The role of health-beneficial lactobacilli prevalent in the human small intestine in starch metabolism remains unexplored in contrast to colonic bacterial residents. This study highlights the pivotal role of debranching enzymes in the breakdown of starchy branched α-glucan oligomers (α-limit dextrins) by human gut lactobacilli exemplified by Lactobacillus acidophilus NCFM, which is one of the best-characterized strains used as probiotics. Our data bring novel insight into the metabolic preference of L. acidophilus for α-glucans with short α-1,6-branches. The unprecedented affinity of the debranching enzyme that confers growth on these substrates reflects its adaptation to the nutrient-competitive gut ecological niche and constitutes a potential advantage in cross-feeding from human and bacterial dietary starch metabolism.

scholarly journals Structure and function of Bs164 β-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164

2019 ◽  
Vol 295 (13) ◽  
pp. 4316-4326 ◽  
Author(s):  
Zachary Armstrong ◽  
Gideon J. Davies
Keyword(s):  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Glycoside Hydrolase Family ◽  
Human Gut ◽  
X Ray Crystallography ◽  
Founding Member ◽  
And Function ◽  
Protein Sequence Space ◽  
Kinetics Of ◽  
Hydrolase Family

Recent work exploring protein sequence space has revealed a new glycoside hydrolase (GH) family (GH164) of putative mannosidases. GH164 genes are present in several commensal bacteria, implicating these genes in the degradation of dietary glycans. However, little is known about the structure, mechanism of action, and substrate specificity of these enzymes. Herein we report the biochemical characterization and crystal structures of the founding member of this family (Bs164) from the human gut symbiont Bacteroides salyersiae. Previous reports of this enzyme indicated that it has α-mannosidase activity, however, we conclusively show that it cleaves only β-mannose linkages. Using NMR spectroscopy, detailed enzyme kinetics of WT and mutant Bs164, and multiangle light scattering we found that it is a trimeric retaining β-mannosidase, that is susceptible to several known mannosidase inhibitors. X-ray crystallography revealed the structure of Bs164, the first known structure of a GH164, at 1.91 Å resolution. Bs164 is composed of three domains: a (β/α)8 barrel, a trimerization domain, and a β-sandwich domain, representing a previously unobserved structural-fold for β-mannosidases. Structures of Bs164 at 1.80–2.55 Å resolution in complex with the inhibitors noeuromycin, mannoimidazole, or 2,4-dinitrophenol 2-deoxy-2-fluoro-mannoside reveal the residues essential for specificity and catalysis including the catalytic nucleophile (Glu-297) and acid/base residue (Glu-160). These findings further our knowledge of the mechanisms commensal microbes use for nutrient acquisition.

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