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A new group of glycoside hydrolase family 13 ?-amylases with an aberrant catalytic triad.

ABSTRACT: ?-Amylases are glycoside hydrolase enzymes that act on the ?(1?4) glycosidic linkages in glycogen, starch, and related ?-glucans, and are ubiquitously present in Nature. Most ?-amylases have been classified in glycoside hydrolase family 13 with a typical (?/?)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical ?-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel ?-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 ?-amylase is part of a group of ?-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of ?-amylases comprises a new subfamily of the glycoside hydrolase family 13.

SUBMITTER: Sarian FD

PROVIDER: S-EPMC5347038 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad.

Sarian Fean D FD Janeček Štefan Š Pijning Tjaard T Ihsanawati Nurachman Zeily Z Radjasa Ocky K OK Dijkhuizen Lubbert L Natalia Dessy D van der Maarel Marc J E C MJ

Scientific reports 20170313

α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)<sub>8</sub>-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillu ...[more]

PMID: 28287181

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Project description:Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using NfEG12A from Neosartorya fischeri P1 and EG (PDB 1KS4) from Aspergillus niger Acidophilic and thermophilic NfEG12A had the highest catalytic efficiency (kcat/Km , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into NfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (kcat/Km ) of these enzymes (in descending order, NfEG12A [100%], NfEG12A-FN [140%], and NfEG12A-STTQA [190%]; EG [100%] and EGΔSTTQA [41%]). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (kcat). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis.IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the -1 or +1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (kcat). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.

Dataset Information

A new group of glycoside hydrolase family 13 ?-amylases with an aberrant catalytic triad.

Publications

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad.

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