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

ABSTRACT 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.

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An 1,4-α-glucosyltransferase defines a new maltodextrin catabolism scheme in Lactobacillus acidophilus

10.1101/2020.03.17.996314 ◽

2020 ◽

Author(s):

Susan Andersen ◽

Marie S. Møller ◽

Jens-Christian N. Poulsen ◽

Michael J. Pichler ◽

Birte Svensson ◽

...

Keyword(s):

Small Intestine ◽

Lactobacillus Acidophilus ◽

Glycoside Hydrolase ◽

Fine Tuning ◽

Substrate Affinity ◽

Concerted Action ◽

Sequence Analyses ◽

Human Small Intestine ◽

Maltose Phosphorylase ◽

Catabolism Pathway

ABSTRACTThe maltooligosaccharide (MOS) utilization locus in Lactobacillus acidophilus NCFM, a model for human small-intestine lactobacilli, encodes a family 13 subfamily 31 glycoside hydrolase (GH13_31), annotated as an 1,6-α-glucosidase. Here, we reveal that this enzyme (LaGH13_31B) is an 1,4-α-glucosyltransferase that disproportionates MOS with preference for maltotriose. LaGH13_31B acts in concert with a maltogenic α-amylase that efficiently releases maltose from MOS larger than maltotriose. Collectively, these two enzymes promote efficient conversion of preferentially odd-numbered MOS to maltose that is phosphorolysed by a maltose phosphorylase, encoded by the same locus. Structural analyses revealed the presence of a flexible elongated loop, which is unique for LaGH13_31B and its close homologues. The identified loop insertion harbours a conserved aromatic residue that modulates the activity and substrate affinity of the enzyme, thereby offering a functional signature of this previously undescribed clade, which segregates from described activities such as 1,6-α-glucosidases and sucrose isomerases within GH13_31. Sequence 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.IMPORTANCEThe 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 human small intestine and considered as beneficial to health. This study unveils a previously unknown scheme of maltooligosaccharide (MOS) catabolism, via the concerted action of activity together with a classical hydrolase and a phosphorylase. The intriguing involvement of a glucosyltransferase is likely to allow fine-tuning 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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00402-17 ◽

2017 ◽

Vol 83 (12) ◽

Cited By ~ 13

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.

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Electron probe x-ray microanalysis and electron microscopy of amino acid absorption and transport by the small intestine: inhibition by chemical poisons and correlation to the elemental composition of the epithelial cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142311 ◽

1986 ◽

Vol 44 ◽

pp. 134-135

Author(s):

A. J. Tousimis

Keyword(s):

Small Intestine ◽

Amino Acid ◽

Epithelial Cells ◽

Elemental Composition ◽

Isotope Labeling ◽

Structural Components ◽

X Ray ◽

Human Small Intestine ◽

Transport Studies

The elemental composition of amino acids is similar to that of the major structural components of the epithelial cells of the small intestine and other tissues. Therefore, their subcellular localization and concentration measurements are not possible by x-ray microanalysis. Radioactive isotope labeling: I131-tyrosine, Se75-methionine and S35-methionine have been successfully employed in numerous absorption and transport studies. The latter two have been utilized both in vitro and vivo, with similar results in the hamster and human small intestine. Non-radioactive Selenomethionine, since its absorption/transport behavior is assumed to be the same as that of Se75- methionine and S75-methionine could serve as a compound tracer for this amino acid.

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