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PUL0230 |
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22685275, J Bacteriol. 2012 Aug;194(16):4249-59. doi: 10.1128/JB.00622-12. Epub 2012 Jun 8. |
| | Enzymology and structure of the GH13_31 glucan 1,6-alpha-glucosidase that confers isomaltooligosaccharide utilization in the probiotic Lactobacillus acidophilus NCFM. |
| | Moller MS, Fredslund F, Majumder A, Nakai H, Poulsen JC, Lo Leggio L, Svensson B, Abou Hachem M |
| | 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 alpha-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 alpha-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 alpha-1,6- and alpha-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-alpha-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 A 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. |
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32444471, Appl Environ Microbiol. 2020 Jul 20;86(15):e00661-20. doi: 10.1128/AEM.00661-20. Print 2020 Jul 20. |
| | An 1,4-alpha-Glucosyltransferase Defines a New Maltodextrin Catabolism Scheme in Lactobacillus acidophilus. |
| | Andersen S, Moller MS, Poulsen JN, Pichler MJ, Svensson B, Lo Leggio L, Goh YJ, Abou Hachem M |
| | The maltooligosaccharide (MOS) utilization locus in Lactobacillus acidophilus NCFM, a model for human small-intestine lactobacilli, encodes three glycoside hydrolases (GHs): a putative maltogenic alpha-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-alpha-glucosidase. Here, we reveal that LaGH13_31B is a 1,4-alpha-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 k(cat) 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-alpha-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 alpha-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-alpha-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-alpha-glucosyl transferases that have been recruited in the MOS catabolism pathway in lactobacilli. |
