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A novel GH13 subfamily of α-amylases with a pair of tryptophans in the helix α3 of the catalytic TIM-barrel, the LPDlx signature in the conserved sequence region V and a conserved aromatic motif at the C-terminus

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Abstract

The α-amylase enzyme specificity has been classified in the Carbohydrate-Active enZyme (CAZy) database into the families GH13, GH57, GH119 and eventually also GH126. α-Amylase is a glycoside hydrolase (GH) that catalyses in an endo-fashion the hydrolysis of the α-1,4-glucosidic linkages in starch and related α-glucans employing the retaining reaction mechanism. The family GH13 is the main a-amylase family with more than 28,000 members and 30 different specificities. The entire family GH13 has already been divided into 40 subfamilies; the a-amylase enzyme specificity being found in the subfamilies GH13_1, 5, 6, 7, 15, 19, 24, 27, 28, 32, 36 and 37. The present in silico study delivers a proposal to create a novel GH13 subfamily with the specificity of a-amylase. The proposal is based on a detailed bioinformatics analysis consisting of sequence, structural and evolutionary comparison of experimentally characterized a-amylases from, e.g., Bacillus aquimaris, Anoxybacillus sp. SK3-4 and DT3-1 and Geobacillus thermoleovorans, and hypothetical proteins, accompanied by a-amylases from well-established GH13 subfamilies and by closely related amylolytic enzymes (mainly from the subfamily GH13_31). Three sequence-structural features can be ascribed to the members of the newly proposed GH13 subfamily: (i) the pair of adjacent tryptophan residues positioned between the CSR-V and CSR-II in the helix a3 of the catalytic TIM-barrel; (ii) the sequence LPDlx in their CSR-V; and (iii) a ~30-residue long C-terminal region with a motif of five conserved aromatic residues. From the evolutionary point of view, the novel GH13 a-amylase subfamily is most closely related to fungal and yeast a-amylases classified in the subfamily GH13_1.

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Abbreviations

BaqA:

Bacillus aquimaris a-amylase

CAZy:

Carbohydrate-Active enZymes

CSR:

conserved sequence region

GH:

glycoside hydrolase

PDB:

Protein Data Bank

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  1. Laboratory of Protein Evolution, Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551, Bratislava, Slovakia

    Štefan Janeček, Andrea Kuchtová & Soňa Petrovičová

  2. Department of Biology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, SK-91701, Trnava, Slovakia

    Štefan Janeček & Soňa Petrovičová

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  1. Štefan Janeček
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  2. Andrea Kuchtová
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Correspondence to Štefan Janeček.

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Janeček, Š., Kuchtová, A. & Petrovičová, S. A novel GH13 subfamily of α-amylases with a pair of tryptophans in the helix α3 of the catalytic TIM-barrel, the LPDlx signature in the conserved sequence region V and a conserved aromatic motif at the C-terminus. Biologia 70, 1284–1294 (2015). https://doi.org/10.1515/biolog-2015-0165

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