An 1,4-α-Glucosyltransferase Defines a New Maltodextrin Catabolism Scheme in Lactobacillus acidophilus.

The maltooligosaccharide (MOS) utilization locus in Lactobacillus acidophilus NCFM, a model for human small-intestine lactobacilli, encodes three glycoside hydrolases (GHs): a putative maltogenic α-amylase of family 13, subfamily 20 (LaGH13_20), a maltose phosphorylase of GH65 (LaGH65), and a family 13, subfamily 31, member (LaGH13_31B), annotated as a 1,6-α-glucosidase. Here, we reveal that LaGH13_31B is a 1,4-α-glucosyltransferase that disproportionates MOS with a degree of polymerization of ≥2, with a preference for maltotriose. Kinetic analyses of the three GHs encoded by the MOS locus revealed that the substrate preference of LaGH13_31B toward maltotriose complements the ~40-fold lower kcat of LaGH13_20 toward this substrate, thereby enhancing the conversion of odd-numbered MOS to maltose. The concerted action of LaGH13_20 and LaGH13_31B confers the efficient conversion of MOS to maltose that is phosphorolyzed by LaGH65. Structural analyses revealed the presence of a flexible elongated loop that is unique for a previously unexplored clade of GH13_31, represented by LaGH13_31B. The identified loop insertion harbors a conserved aromatic residue that modulates the activity and substrate affinity of the enzyme, thereby offering a functional signature of this clade, which segregates from 1,6-α-glucosidases and sucrose isomerases previously described within GH13_31. Genomic analyses revealed that the LaGH13_31B gene is conserved in the MOS utilization loci of lactobacilli, including acidophilus cluster members that dominate the human small intestine.IMPORTANCE The degradation of starch in the small intestine generates short linear and branched α-glucans. The latter are poorly digestible by humans, rendering them available to the gut microbiota, e.g., lactobacilli adapted to the small intestine and considered beneficial to health. This study unveils a previously unknown scheme of maltooligosaccharide (MOS) catabolism via the concerted activity of an 1,4-α-glucosyltransferase together with a classical hydrolase and a phosphorylase. The intriguing involvement of a glucosyltransferase likely allows the fine-tuning of the regulation of MOS catabolism for optimal harnessing of this key metabolic resource in the human small intestine. The study extends the suite of specificities that have been identified in GH13_31 and highlights amino acid signatures underpinning the evolution of 1,4-α-glucosyl transferases that have been recruited in the MOS catabolism pathway in lactobacilli.

A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan.

Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 µm and that it does not bind to starch mimics, ß-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a ß-d-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1-CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.

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

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 ß-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.

Crystal structure of barley limit dextrinase-limit dextrinase inhibitor (LD-LDI) complex reveals insights into mechanism and diversity of cereal type inhibitors.

Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.

Oligosaccharide and substrate binding in the starch debranching enzyme barley limit dextrinase.

Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD-substrate interactions that confine high activity of LD to branched maltooligosaccharides. For the first time, an intact α-1,6-glucosidically linked substrate spanning the active site of a LD or pullulanase has been trapped and characterized by crystallography. The crystal structure reveals both the branch and main-chain binding sites and is used to suggest a mechanism for nucleophilicity enhancement in the active site. The substrate, product and analogue complexes were further used to outline substrate binding subsites and substrate binding restraints and to suggest a mechanism for avoidance of dual α-1,6- and α-1,4-hydrolytic activity likely to be a biological necessity during starch synthesis.

Enzymology and structure of the GH13_31 glucan 1,6-α-glucosidase that confers isomaltooligosaccharide utilization in the probiotic Lactobacillus acidophilus NCFM.

Isomaltooligosaccharides (IMO) have been suggested as promising prebiotics that stimulate the growth of probiotic bacteria. Genomes of probiotic lactobacilli from the acidophilus group, as represented by Lactobacillus acidophilus NCFM, encode α-1,6 glucosidases of the family GH13_31 (glycoside hydrolase family 13 subfamily 31) that confer degradation of IMO. These genes reside frequently within maltooligosaccharide utilization operons, which include an ATP-binding cassette transporter and α-glucan active enzymes, e.g., maltogenic amylases and maltose phosphorylases, and they also occur separated from any carbohydrate transport or catabolism genes on the genomes of some acidophilus complex members, as in L. acidophilus NCFM. Besides the isolated locus encoding a GH13_31 enzyme, the ABC transporter and another GH13 in the maltooligosaccharide operon were induced in response to IMO or maltotetraose, as determined by reverse transcription-PCR (RT-PCR) transcriptional analysis, suggesting coregulation of α-1,6- and α-1,4-glucooligosaccharide utilization loci in L. acidophilus NCFM. The L. acidophilus NCFM GH13_31 (LaGH13_31) was produced recombinantly and shown to be a glucan 1,6-α-glucosidase active on IMO and dextran and product-inhibited by glucose. The catalytic efficiency of LaGH13_31 on dextran and the dextran/panose (trisaccharide) efficiency ratio were the highest reported for this class of enzymes, suggesting higher affinity at distal substrate binding sites. The crystal structure of LaGH13_31 was determined to a resolution of 2.05 Å and revealed additional substrate contacts at the +2 subsite in LaGH13_31 compared to the GH13_31 from Streptococcus mutans (SmGH13_31), providing a possible structural rationale to the relatively high affinity for dextran. A comprehensive phylogenetic and activity motif analysis mapped IMO utilization enzymes from gut microbiota to rationalize preferential utilization of IMO by gut residents.