Identification of Catalytic and Substrate-binding Site Residues in Bacillus cereus ATCC7064 Oligo-1,6-glucosidase

Watanabe, Kunihiko; Miyake, Kazue; Suzuki, Yuzuru

doi:10.1271/bbb.65.2058

Cited by 31 publications

(17 citation statements)

References 26 publications

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“…Remarkably, utilization of IMO by human gut bacteria and especially lactobacilli is largely unexplored on the enzymatic level as no IMO-degrading enzymes from a probiotic have been characterized to date. By comparison, Gram-positive bacteria from the Streptococcus and Bacillus genera are known to produce 1,6-␣-glucosidases active on IMO and in some cases also on dextran (␣-1,6-linked glucan) (43,47,54,55). These enzymes hydrolyze ␣-1,6-glycosidic linkages at the nonreducing end with retention of anomeric configuration (EC 3.2.1.10) and belong to glycoside hydrolase family 13 subfamily 31 (GH13_31) (46) according to the CAZy database classification (http://www.cazy.org/), which assigns carbohydrate-active enzymes into GH families sharing structural fold and stereochemical mechanisms (8) (sequence-and structure-related families are further classified into clans).…”

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“…The latter are divided into two specificities based on substrate size preference: (i) glucan 1,6-␣-glucosidases (G16G) preferring IMO longer than IG2 and active on dextran and (ii) oligo-1,6-␣-glucosidases (O16G) inactive on dextran and preferring shorter IMO with highest activity on IG2. Both types of enzymes are highly regioselective, showing little or no activity toward glucosidic linkages other than ␣-1,6 (43,47,54,55). The only biochemically and structurally characterized GH13_31 with a glucan 1,6-␣-glucosidase specificity (G16G) is from Streptococcus mutans (SmGH13_31) (19,43).…”

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confidence: 99%

“…The only biochemically and structurally characterized GH13_31 with a glucan 1,6-␣-glucosidase specificity (G16G) is from Streptococcus mutans (SmGH13_31) (19,43). By comparison, the biochemical properties of three GH13_31 oligo-1,6-␣-glucosidases (O16Gs) from Bacillus thermoglucosidasius (Geobacillus thermoglucosidasius), Bacillus coagulans, and Bacillus cereus have been reported, and the structure of the last enzyme has been determined (43,47,54,55).…”

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

et al. 2012

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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.

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

et al. 2012

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“…GmdH also liberates glucose from isomaltose and isomaltotriose. Its sequence and substrate specificity strongly resemble those of ␣-1,6-glucosidases in B. subtilis (YcdG), Bacillus cereus (26), and Bacillus coagulans (27).…”

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confidence: 98%

Enzymes Required for Maltodextrin Catabolism in Enterococcus faecalis Exhibit Novel Activities

Joyet

Mokhtari

Riboulet-Bisson

et al. 2017

Appl Environ Microbiol

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Maltose and maltodextrins are formed during the degradation of starch or glycogen. Maltodextrins are composed of a mixture of maltooligosaccharides formed by ␣-1,4-but also some ␣-1,6-linked glucosyl residues. The ␣-1,6-linked glucosyl residues are derived from branching points in the polysaccharides. In Enterococcus faecalis, maltotriose is mainly transported and phosphorylated by a phosphoenolpyruvate:carbohydrate phosphotransferase system. The formed maltotriose-6Љ-phosphate is intracellularly dephosphorylated by a specific phosphatase, MapP. In contrast, maltotetraose and longer maltooligosaccharides up to maltoheptaose are taken up without phosphorylation via the ATP binding cassette transporter MdxEFG-MsmX. We show that the maltose-producing maltodextrin hydrolase MmdH (GenBank accession no. EFT41964) in strain JH2-2 catalyzes the first catabolic step of ␣-1,4-linked maltooligosaccharides. The purified enzyme converts even-numbered ␣-1,4-linked maltooligosaccharides (maltotetraose, etc.) into maltose and odd-numbered (maltotriose, etc.) into maltose and glucose. Inactivation of mmdH therefore prevents the growth of E. faecalis on maltooligosaccharides ranging from maltotriose to maltoheptaose. Surprisingly, MmdH also functions as a maltogenic ␣-1,6-glucosidase, because it converts the maltotriose isomer isopanose into maltose and glucose. In addition, E. faecalis contains a glucose-producing ␣-1,6-specific maltodextrin hydrolase (GenBank accession no. EFT41963, renamed GmdH). This enzyme converts panose, another maltotriose isomer, into glucose and maltose. A gmdH mutant had therefore lost the capacity to grow on panose. The genes mmdH and gmdH are organized in an operon together with GenBank accession no. EFT41962 (renamed mmgT). Purified MmgT transfers glucosyl residues from one ␣-1,4-linked maltooligosaccharide molecule to another. For example, it catalyzes the disproportionation of maltotriose by transferring a glucosyl residue to another maltotriose molecule, thereby forming maltotetraose and maltose together with a small amount of maltopentaose.IMPORTANCE The utilization of maltodextrins by Enterococcus faecalis has been shown to increase the virulence of this nosocomial pathogen. However, little is known about how this organism catabolizes maltodextrins. We identified two enzymes involved in the metabolism of various ␣-1,4-and ␣-1,6-linked maltooligosaccharides. We found that one of them functions as a maltose-producing ␣-glucosidase with relaxed linkage specificity (␣-1,4 and ␣-1,6) and exo-and endoglucosidase activities. A third enzyme, which resembles amylomaltase, exclusively transfers glucosyl residues from one maltooligosaccharide molecule to another. Similar enzymes are present in numerous other Firmicutes, such as

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“…They are usually found in association with other amylolytic enzymes which accomplish complete degradation of starch, and are widely distrubuted throughout the three major kingdoms and occur in microorganisms as intracellular, extracellular, or cell-bound enzymes (Kelly and Fogarty 1983;Watanabe et al 2001). a-Glucosidases, responsible for hydrolysis of p-nitrophenol-a-D-glucopyranoside (pNPG), have also been found in a number of different thermophilic Bacillus species (Suzuki et al 1984).…”

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Characterization of thermostable α-glucosidases from newly isolated Geobacillus sp. A333 and thermophilic bacterium A343

Cihan

Özcan

Çökmüş

2009

World J Microbiol Biotechnol

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We have partially purified and characterized two new thermostable exo-a-1,4-glucosidases (E.C.3.2.1.20) isolated from Geobacillus sp. A333 and thermophilic bacterium A343 strains. A333 a-glucosidase showed optimum activity at 60°C, pH 6.8 and had a value of 1.38 K m for the pNPG substrate, whereas these results were found to be 65°C, 7.0 and 0.85, respectively for A343 enzyme. Specificity for 20 different substrates and thin layer chromatography studies demonstrated that the A333 enzyme had high transglycosylation activity, and A343 had wide substrate specificity. The substrate specificity of A333 a-glucosidase was determined as maltose, dextrin, turanose, maltotriose, maltopentaose, meltotetraose, maltohexaose and phenyla-D-glycopyranoside. On the other hand, the A343 a-glucosidase mostly hydrolyzed dextrin, turanose, maltose, phenyl-a-D-glucopyranoside, maltotriose, maltotetraose, maltopentaose, isomaltose, saccharose and kojibiose by acting a-1,2, a-1,3, a-1,4 and a-1,6 bonds of these substrates. The relative activites of A333 and A343 enzymes were determined to be 83 and 92% when incubated at 60°C for 5 h whereas, the pH of 50% inactivation at 60°C for 15 h were determined to be pH 4.5/10.0 and pH 5.0/10.0, respectively. In addition, the results not only showed that both of the a-glucosidases were stable in a wide range of pH and temperatures, but were also found to be resistant to most of the denaturing agents, inhibitors and metal ions tested. With this study, thermostable exo-a-1,4-glucosidases produced by two new thermophilic strains were characterized as having biotechnological potential in transglycosylation reactions and starch hydrolysis processes.

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Identification of Catalytic and Substrate-binding Site Residues in Bacillus cereus ATCC7064 Oligo-1,6-glucosidase

Cited by 31 publications

References 26 publications

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

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

Enzymes Required for Maltodextrin Catabolism in Enterococcus faecalis Exhibit Novel Activities

Characterization of thermostable α-glucosidases from newly isolated Geobacillus sp. A333 and thermophilic bacterium A343

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