Function and structure of <scp>GH</scp>13_31 α‐glucosidase with high α‐(1→4)‐glucosidic linkage specificity and transglucosylation activity

Auiewiriyanukul, Waraporn; Saburi, Wataru; Kato, Kuniki; Yao, Min; Mori, Haruhide

doi:10.1002/1873-3468.13126

Cited by 35 publications

(48 citation statements)

References 46 publications

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“…2A). A Ca 2+ binding site formed by side chains of D20, N22, D24, D28 and main chain carbonyls of I26 and H73, is identified in a similar location to counterparts present in some GH13_31 structures (22).…”

Section: Resultsmentioning

confidence: 67%

“…3A), the recently characterised α-glucosidase from Bacillus sp. Bsp AG13_31A (22) and a Geobacillus α-glucosidase (23) were found to be the closest structurally characterized enzymes.…”

Section: Resultsmentioning

confidence: 99%

“…S5). Notably, the corresponding but shorter loop in Bsp AG13_31A was suggested to be flexible in Bsp AG13_31A based on the the poor electron density of the loop and ligand-binding dependent conformational changes in this loop (22).…”

Section: Resultsmentioning

confidence: 99%

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

Andersen¹,

Moeller²,

Poulsen

et al. 2020

Preprint

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The 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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Section: Resultsmentioning

confidence: 67%

“…3A), the recently characterised α-glucosidase from Bacillus sp. Bsp AG13_31A (22) and a Geobacillus α-glucosidase (23) were found to be the closest structurally characterized enzymes.…”

Section: Resultsmentioning

confidence: 99%

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

Andersen¹,

Moeller²,

Poulsen

et al. 2020

Preprint

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“…LaGH13_31B shares the catalytic machinery and the overall domain structure of amylolytic GH13 enzymes, namely a (β/α)8 catalytic domain A (residues 1-477; catalytic residues D198, E255, and D334) with two inserted domains (domain B, residues 100-169; domain B′, residues 373-459), and a domain C composed primarily of β-sheets (478-550) ( Fig. 2A) (22).…”

Section: Lagh13_31b Displays a More Open Active Site With A Potentialmentioning

confidence: 99%

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

Andersen

Møller

Poulsen

et al. 2020

Appl Environ Microbiol

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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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“…Group I includes the ones that hydrolyse heterogeneous substrates (e.g. sucrose and aryl-glucosides) more efficiently than homogeneous substrates as maltose, the group II are more active on homogeneous substrates as maltose and isomaltose and finally, the group III comprise enzymes that exhibit the same activity as the type II, but also hydrolyse long-chain substrates [16,18]. Based on a structural classification, α-glucosidases are distributed into five glycoside hydrolase (GH) families: GH4, GH13, GH31, GH63 and GH97 [19].…”

Section: Introductionmentioning

confidence: 99%

Molecular characterization and heterologous expression of two α-glucosidases from Metschnikowia spp, both producers of honey sugars

et al. 2020

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Background: α-Glucosidases are widely distributed enzymes with a varied substrate specificity that are traditionally used in biotechnological industries based on oligo-and polysaccharides as starting materials. According to amino acid sequence homology, α-glucosidases are included into two major families, GH13 and GH31. The members of family GH13 contain several α-glucosidases with confirmed hydrolytic activity on sucrose. Previously, a sucrose splitting activity from the nectar colonizing yeast Metschnikowia reukaufii which produced rare sugars with α-(1→1), α-(1→3) and α-(1→6) glycosidic linkages from sucrose was described. Results: In this study, genes codifying for α-glucosidases from the nectaries yeast M. gruessii and M. reukaufii were characterised and heterologously expressed in Escherichia coli for the first time. Recombinant proteins (Mg-αGlu and Mr-αGlu) were purified and biochemically analysed. Both enzymes mainly displayed hydrolytic activity towards sucrose, maltose and p-nitrophenyl-α-d-glucopyranoside. Structural analysis of these proteins allowed the identification of common features from the α-amylase family, in particular from glycoside hydrolases that belong to family GH13. The three acidic residues comprising the catalytic triad were identified and their relevance for the protein hydrolytic mechanism confirmed by site-directed mutagenesis. Recombinant enzymes produced oligosaccharides naturally present in honey employing sucrose as initial substrate and gave rise to mixtures with the same products profile (isomelezitose, trehalulose, erlose, melezitose, theanderose and esculose) previously obtained with M. reukaufii cell extracts. Furthermore, the same enzymatic activity was detected with its orthologous Mg-αGlu from M. gruessii. Interestingly, the isomelezitose amounts obtained in reactions mediated by the recombinant proteins, ~ 170 g/L, were the highest reported so far. Conclusions: Mg/Mr-αGlu were heterologously overproduced and their biochemical and structural characteristics analysed. The recombinant α-glucosidases displayed excellent properties in terms of mild reaction conditions, in addition to pH and thermal stability. Besides, the enzymes produced a rare mixture of hetero-gluco-oligosaccharides by transglucosylation, mainly isomelezitose and trehalulose. These compounds are natural constituents of honey which purification from this natural source is quite unviable, what make these enzymes very interesting for the

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Function and structure of GH13_31 α‐glucosidase with high α‐(1→4)‐glucosidic linkage specificity and transglucosylation activity

Cited by 35 publications

References 46 publications

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

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

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

Molecular characterization and heterologous expression of two α-glucosidases from Metschnikowia spp, both producers of honey sugars

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