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.

Niche-specific adaptation of Lactobacillus helveticus strains isolated from malt whisky and dairy fermentations

“…The genes encoding the two enzymes were located adjacently in the genome and were next to a gene encoding GH65-1 maltose phosphorylase, resulting in the formation of PUL-GH13-GH65 in the milk strain CNRZ32. Similar PUL organization was observed in several species in the L. acidophilus group [ 35 ]. The PUL was characterized by the release of maltose from maltooligosaccharides through the cooperation of two GH13 enzymes and further phosphorylation of maltose into β- d -glucose 1-phosphate and glucose by the GH65 enzyme in L. acidophilus .…”

“…Similar PUL organization was observed in several species in the L. acidophilus group [35]. The PUL was characterized by the release of maltose from maltooligosaccharides through the cooperation of two GH13 enzymes and further phosphorylation of maltose into β- d -glucose 1-phosphate and glucose by the GH65 enzyme in L.…”

“…However, clusters 3 (GH13-3), 4 (GH13-4) and 8 (GH13-8) were composed of proteins only found in certain milk strains, whereas the strains included differed among the clusters. Oligo-1,6-α-glucosidase and 1,4-α-glucosyltransferase of L. acidophilus NCFM, which cooperatively function in the hydrolysis of maltooligosaccharides/starch [ 35 ], belong to cluster 5 (GH13-5) and cluster 7 (GH13-7), respectively. GH13-5 and GH13-7 proteins were found in all whisky strains and 8 of the 14 milk strains.…”

“…GH13-5 and GH13-7 included reference maltooligosaccharide/starch degrading enzymes of L. acidophilus [ 35 ]. The genes encoding the two enzymes were located adjacently in the genome and were next to a gene encoding GH65-1 maltose phosphorylase, resulting in the formation of PUL-GH13-GH65 in the milk strain CNRZ32.…”

Oligosaccharide Metabolism and Lipoteichoic Acid Production in Lactobacillus gasseri and Lactobacillus paragasseri

“…A large gene cluster (gene IDs ranging from 14610 to 14690), containing genes encoding three GH13 enzymes and GH65, was highly transcribed in the presence of glucose and raffinose when compared with kestose, and transcription levels of most of these genes were similar between cells cultured with glucose and raffinose. The gene cluster was characterized by the degradation of starch and maltooligosaccharides in a phylogenetically related taxon, Lactobacillus acidophilus [ 38 ], and was not used for the metabolism of glucose or raffinose. This suggests that the different transcription levels observed in the present study were due to repression of these genes by kestose.…”