Enzymology and Structure of the GH13_31 Glucan 1,6-α-Glucosidase That Confers Isomaltooligosaccharide Utilization in the Probiotic Lactobacillus acidophilus NCFM

Abstract

b 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 ␣-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 ␣-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 ␣-1,6-and ␣-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-␣-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.

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

“…The commercial and physiological relevance of L. acidophilus NCFM has spurred wide interest in the saccharide uptake and catabolism machinery of this organism to identify efficiently utilized glycans with potential as prebiotics (25,38,39). A few intracellular carbohydrate-active enzymes (CAZymes) from L. acidophilus NCFM have been characterized, including those active on maltose (26) and ␣-1,6-linked isomalto-oligosaccharides (24). However, insight is scarce into the extracellular CAZymes encoded by this bacterium and related human-gut-adapted lactobacilli (27).…”

“…The poor growth on maltotriose and maltotetraose is, however, surprising as these substrates are predicted to be internalized through a maltodextrin-specific ATP-binding cassette (ABC) system (LBA1864 to LBA1867), which is conserved in acidophilus group lactobacilli (24). The corresponding transporter from the taxonomically related Gram-positive pathogen Streptococcus pneumoniae mediates the uptake of malto-oligosaccharides up to eight units with a preference for maltotetraose (44).…”

Recent insight in α-glucan metabolism in probiotic bacteria

“…1A). This subfamily contains α-1,6-glucosidases that confer the utilization of α-1,6-containing gluco-oligosaccharides (IMOs and panose), consistent with the prebiotic effect of this type of glucans that endow probiotic bacteria with a competitive edge due to their preferential fermentation (Kaneko et al 1994;Yen et al 2011;Møller et al 2012). Other subfamilies that are highly represented in both genera are GH13 18 sucrose phosphorylases and GH13 9 1,4-α-glucan branching enzymes (Fig.…”

“…This arrangement is highly conserved across species in the genus with the main difference being that in other species, the gene cluster also includes a GH13 31 glucan α-1,6-glucosidase that catalyses the hydrolysis of IMOs including panose. Recently, the glucan α-1,6-glucosidase from L. acidophilus NCFM, which is encoded by a separate genetic locus than the malto-oligosaccharide operon, has been biochemically and structurally characterized providing the first insight into the enzymology α-1,6-glucosidases from lactobacilli (Møller et al 2012). The frequent colocalization of α-1,6-glucosidase genes in the maltooligosaccharide operon in most lactobacilli raises the question of whether the same ABC transport system is able to confer uptake of both IMOs and maltooligosaccharides, but experimental evidence is needed to verify this.…”

“…The former subfamily confers the phosphorolysis of sucrose moieties including those derived from soybean oligosaccharides of the raffinose family, which are also suggested to be prebiotics (Fredslund et al 2011;Andersen et al 2012), whereas the branching enzymes of the latter subfamily GH13 9 are involved in glycogen synthesis . Some subfamilies are uniquely encountered in Lactobacillus, e.g., GH13 20 maltogenic α-amylases that are frequently present in the malto-oligosaccharide metabolism operons in lactobacilli (Nakai et al 2009;Møller et al 2012), while others are unique for bifidobacteria, e.g., predicted extracellular α-amylases of GH13 32 and modular amylopullulanases containing both GH13 14 and GH13 32 catalytic modules (Fig. 1A, Fig.…”