Probing the Substrate Specificity and Intersubunit Interactions of Brevundimonas Diminuta Glutaryl Acylase with Site-Directed Mutagenesis
- 1 Russian Academy of Sciences, Russia
- 2 Lomonosov Moscow State University, Russia
Abstract
Glutaryl-7-ACA acylases (GLA) are industrial enzymes widely used for the production of 7-aminocepahlosporanic acid (7-ACA)-the starting compound for manufacturing of semisynthetic cephalosporin antibiotics. Generation of mutant GLA's with increased activity and stability, capability for single-step conversion of cephalosporin C (CPC) directly to 7-ACA is a promising route to improve the current biocatalytic technologies. In this study GLA from Brevundimonas diminuta (BrdGLA) has been rationally re-designed to produce enzyme variants with improved properties. First, sequence analysis was performed to select residues responsible for substrate specificity as hotspots to introduce the capability to bind CPC in the active site of BrdGLA. Molecular modeling was used to evaluate the influence of selected residues on the formation of productive enzyme-substrate complex and the catalytic conversion. Genes, encoding mutant enzymes, were constructed, expressed and recombinant enzymes were purified on the chitin affinity resin. The mutant proteins showed induced catalytic activity against CPC. Second, BrdGLA mutants with increased activity and stability in alkaline conditions were obtained by mutating one of the surface lysine residues and replacement of the glutamine residue located in the active center by asparagine. Finally, structural analysis was used to select amino acid residues involved in formation of the quaternary structure of BrdGLA. Replacement of these "interface" positions to alanines led to a significant enzyme destabilization and reduction of its activity, confirming the role of the identified residues in the intersubunit interactions. The glutaraldehyde cross-linking has shown that the wild-type enzyme and its "interface" mutants possess complex oligomeric structure in solution with predominance of tetrameric forms.
DOI: https://doi.org/10.3844/ajbbsp.2014.169.179
Copyright: © 2014 Michael Eldarov, Anna Lyashenko, Tatyana Sherbakova, Dmitry Suplatov, KirillKopylov and Vytas Svedas. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Keywords
- Cephalosporin C
- Biocatalysis
- Heterologous Expression
- Molecular Modeling
- Site-Directed Mutagenesis