Dataset Information

Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1-2 by Directed Evolution.

ABSTRACT: The glycoside hydrolase LXYL-P1-2 from Lentinula edodes can specifically hydrolyze 7-?-xylosyltaxanes to form 7-?-hydroxyltaxanes for the semi-synthesis of paclitaxel. In order to improve the catalytic properties of the enzyme, we performed error-prone PCR to construct the random mutant library of LXYL-P1-2 and used the methanol-induced plate method to screen the mutants with improved catalytic properties. Two variants, LXYL-P1-2-EP1 (EP1, S91D mutation) and LXYL-P1-2-EP2 (EP2, T368E mutation), were obtained from the library and exhibited 17% and 47% increases in their catalytic efficiencies on 7-?-xylosyl-10-deacetyltaxol. Meanwhile, compared with LXYL-P1-2, EP1 and EP2 showed elevated stabilities in the range of pH ? 6 conditions. After treatment at pH 12 for 48 h, EP1 and EP2 retained 77% and 63% activities, respectively, while the wild-type only retained 33% activity under the same condition. Molecular docking results revealed that the S91D mutation led to a shorter distance between the R-chain and the substrate, while the T368E mutation increased negative charge at the surface of the enzyme, and may introduce alterations of the loop near the active pocket, both of which may result in improved stabilities and catalytic activities of enzymes. This study provides a practical directed evolution method for exploring catalytically improved glycoside hydrolase.

SUBMITTER: Chen JJ

PROVIDER: S-EPMC6149855 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1-2 by Directed Evolution.

Chen Jing-Jing JJ Liang Xiao X Li Hui-Xian HX Chen Tian-Jiao TJ Zhu Ping P

Molecules (Basel, Switzerland) 20171204 12

The glycoside hydrolase LXYL-P1-2 from <i>Lentinula edodes</i> can specifically hydrolyze 7-β-xylosyltaxanes to form 7-β-hydroxyltaxanes for the semi-synthesis of paclitaxel. In order to improve the catalytic properties of the enzyme, we performed error-prone PCR to construct the random mutant library of LXYL-P1-2 and used the methanol-induced plate method to screen the mutants with improved catalytic properties. Two variants, LXYL-P1-2-EP1 (EP1, S91D mutation) and LXYL-P1-2-EP2 (EP2, T368E muta ...[more]

PMID: 29207529

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Project description:BackgroundTrehalases have potential applications in several fields, including food additives, insecticide development, and transgenic plant. In the present study, we focused on a trehalase from the marine bacterium Zunongwangia sp., which hydrolyzes trehalose to glucose.ResultsA novel gene, treZ (1590 bp) encoding an α, α-trehalase of 529 amino acids was cloned from Zunongwangia sp., and TreZ was found to have an optimal activity at 50 °C and pH 6. The activity of TreZ was increased by the presence of NaCl, showing the highest activity (136 %) at 1 M NaCl. A variant C4 with improved catalytic activity was obtained by error-prone PCR and followed by a 96-well plate high-throughput screening. The variant C4 with two altered sites (Y227H, and R442G) displayed a 3.3 fold increase in catalytic efficiency (k cat/K m, 1143.40 mmol(-1) s(-1)) compared with the wild type enzyme (265.91 mmol(-1) s(-1)). In order to explore the contribution of the mutations found in variant C4 to the increased catalytic activity, two mutants Y227H and R442G were constructed by site-directed mutagenesis. The results showed that the catalytic efficiencies of Y227H and R442G were 416.78 mmol(-1) s(-1) and 740.97 mmol(-1) s(-1), respectively, indicating that both mutations contributed to the increased catalytic efficiency of variant C4. The structure modeling and substrate docking revealed that the substitution Y227H enlarged the shape of the binding pocket, to improve the binding of the substrate and the release of the products; while the substitution R442G reduced the size of the side chain and decreased the steric hindrance, which contributed to channel the substrate into the active cavity easier and promote the release of the product.ConclusionIn this study, a novel trehalase was cloned, purified, characterized, and engineered. A variant C4 with dramatically improved catalytic activity was obtained by directed evolution, and the mutation sites Y227H and R442G were found to play a significant role in the catalytic efficiency. The overall results provide useful information about the structure and function of trehalase.

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

Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1-2 by Directed Evolution.

Publications

Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1-2 by Directed Evolution.

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