Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida.
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.
Siegert
Petra
P
Institute of Molecular Enzyme Technology, Heinrich-Heine University of Düsseldorf, Research Centre Jülich, D-52426 Jülich, Germany.
McLeish
Michael J
MJ
Baumann
Martin
M
Iding
Hans
H
Kneen
Malea M
MM
Kenyon
George L
GL
Pohl
Martina
M
D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics*; D011767:Pyruvate Decarboxylase / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity / Q000235:genetics*; D013835:Thiamine Pyrophosphate / Q000378:metabolism; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics*
D002262:Carboxy-Lyases; D011767:Pyruvate Decarboxylase; C019778:benzoylformate decarboxylase; D013835:Thiamine Pyrophosphate