PEDS Advance Access originally published online on June 1, 2005
Protein Engineering Design and Selection 2005 18(7):345-357; doi:10.1093/protein/gzi035
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Published by Oxford University Press 2005
Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida
1Institute of Molecular Enzyme Technology, Heinrich-Heine University of Düsseldorf, Research Centre Jülich, D-52426 Jülich, Germany and 3College of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065, USA 2Present address: Henkel KgaA, Research Technology, Enzyme Technology, Henkelstrasse 67, D-40191 Düsseldorf, Germany 4Present address: Royal Institute of Technology, Alba Nova, Wood-Biotechnology, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden 5Present address: Hoffmann-La Roche AG, Emil-Barell-Strasse 1, D-79639 Grenzach-Wyhlen, Germany
6 To whom correspondence should be addressed. E-mail: ma.pohl{at}fz-juelich.de
Pyruvate decarboxylase from Zymomonas mobilis (PDC) and benzoylformate decarboxylase from Pseudomonas putida (BFD) are thiamine diphosphate-dependent enzymes that decarboxylate 2-keto acids. Although they share a common homotetrameric structure they have relatively low sequence similarity and different substrate spectra. PDC prefers short aliphatic substrates whereas BFD favours aromatic 2-keto acids. These preferences are also reflected in their carboligation reactions. PDC catalyses the conversion of benzaldehyde and acetaldehyde to (R)-phenylacetylcarbinol and predominantly (S)-acetoin, whereas (R)-benzoin and mainly (S)-2-hydroxypropiophenone are the products of BFD catalysis. Comparison of the X-ray structures of both enzymes identified two residues in each that were likely to be involved in determining substrate specificity. Site-directed mutagenesis was used to interchange these residues in both BFD and PDC. The substrate range and kinetic parameters for the decarboxylation reaction were studied for each variant. The most successful variants, PDCI472A and BFDA460I, catalysed the decarboxylation of benzoylformate and pyruvate, respectively, although both variants now preferred the long-chain aliphatic substrates, 2-ketopentanoic and 2-ketohexanoic acid. With respect to the carboligase activity, PDCI472A proved to be a real chimera between PDC and BFD whereas BFDA460I/F464I provided the most interesting result with an almost complete reversal of the stereochemistry of its 2-hydroxypropiophenone product.
Keywords: carboligation/decarboxylation/substrate range/thiamine diphosphate
Received March 25, 2005; revised and accepted May 9, 2005
Edited by Bauke Dijkstra
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