Original Research ARTICLE
Identification of key residues for enzymatic carboxylate reduction
- 1Institute for Molecular Biotechnology, Graz University of Technology, Austria
- 2Austrian Centre of Industrial Biotechnology, Austria
- 3Institute of Molecular Biosciences, University of Graz, Austria
Carboxylate reductases (CARs, E.C. 184.108.40.206) generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzymatic carboxylate reduction.
A multiple sequence alignment of CARs with confirmed activity from the literature and recently identified in our lab revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of Neurospora crassa CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analysing enzymatic activity of the variants in vivo and in vitro. Supported by molecular modelling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of NcCAR variants when replaced by alanine. Finally, we showed that NcCAR wild-type and mutants efficiently reduce aliphatic acids.
Keywords: Carboxylate reductase, Biocatalysis, Signature sequence, Aldehyde, flavour and fragrance, Pharmaceutical intermediate, modelling, Mutation
Received: 23 Nov 2017;
Accepted: 31 Jan 2018.
Edited by:Caroline E. Paul, Wageningen University & Research, Netherlands
Reviewed by:Lígia O. Martins, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal
Peter-Leon Hagedoorn, Delft University of Technology, Netherlands
Copyright: © 2018 Stolterfoht, Steinkellner, Schwendenwein, Pavkov-Keller, Gruber and Winkler. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Dr. Margit Winkler, WINKLER., Austrian Centre of Industrial Biotechnology, Petersgasse 14, Graz, 8010, Austria, email@example.com