Dataset Information

Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering.

ABSTRACT: Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (?Tm ? +3 °C). By combining mutations, a 10-fold mutant was generated with a Tm of 94 °C (+51 °C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a Tm of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.

SUBMITTER: Aalbers FS

PROVIDER: S-EPMC7164962 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering.

Aalbers Friso S FS Fürst Maximilian Jlj MJ Rovida Stefano S Trajkovic Milos M Gómez Castellanos J Rubén JR Bartsch Sebastian S Vogel Andreas A Mattevi Andrea A Fraaije Marco W MW

eLife 20200331

Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (Δ<i>T</i><sub>m</sub> ≥ +3 °C). By combining mutations, a 10-fold mutant was generated ...[more]

PMID: 32228861

Similar Datasets

Project description:We have determined the nucleotide sequences of eight ethyl methanesulphonate-induced mutants in Drosophila alcohol dehydrogenase (ADH), of which six were previously characterized by Hollocher and Place [(1988) Genetics 116, 253-263 and 265-274]. Four of these ADH mutants contain a single amino acid change: glycine-17 to arginine, glycine-93 to glutamic acid, alanine-159 to threonine, and glycine-184 to aspartic acid. Although these mutants are inactive, three mutants (Gly17Arg, Gly93Glu and Gly184Asp) form stable homodimers, as well as heterodimers with wild-type ADH, in which the wild-type ADH subunit retains full enzyme activity [Hollocher and Place (1988) Genetics 116, 265-274]. Interestingly, the Ala159Thr mutant does not form either stable homodimers or heterodimers with wild-type ADH, suggesting that alanine-159 is important in stabilizing ADH dimers. The mutations were analysed in terms of a three-dimensional model of ADH using bacterial 20 beta-hydroxysteroid dehydrogenase and rat dihydropteridine reductase as templates. The model indicates that mutations in glycine-17 and glycine-93 affect the binding of NAD+. It also shows that alanine-159 is part of a hydrophobic anchor on the dimer interface of ADH. Replacement of alanine-159 with threonine, which has a larger side chain and can hydrogen bond with water, is likely to reduce the strength of the hydrophobic interaction. The three-dimensional model shows that glycine-184 is close to the substrate binding site. Replacement of glycine-184 with aspartic acid is likely to alter the position of threonine-186, which we propose hydrogen bonds to the carboxamide moiety of NAD+. Also, the negative charge on the aspartic acid side chain may interact with the substrate and/or residues in the substrate binding site. These mutations provide information about ADH catalysis and the stability of dimers, which may also be useful in understanding homologous dehydrogenases, which include the human 17 beta-hydroxysteroid, 11 beta-hydroxysteroid and 15-hydroxyprostaglandin dehydrogenases.

Dataset Information

Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering.

Publications

Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure