Correspondence to M. Pohl, Institut für Enzymtechnologie der Heinrich-Heine Universität Düsseldorf, Forschungszentrum Jülich, D-52426 Jülich, Germany
Active site mutants of pyruvate decarboxylase from Zymomonas mobilis
A site-directed mutagenesis study of L112, I472, I476, E473 and N482
Article first published online: 25 DEC 2001
European Journal of Biochemistry
Volume 257, Issue 3, pages 538–546, November (I) 1998
How to Cite
Pohl, M., Siegert, P., Mesch, K., Bruhn, H. and Grötzinger, J. (1998), Active site mutants of pyruvate decarboxylase from Zymomonas mobilis. European Journal of Biochemistry, 257: 538–546. doi: 10.1046/j.1432-1327.1998.2570538.x
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Abbreviations. BFD, benzoylformate decarboxylase; ee, enantiomeric excess; 2-HPP, 2-hydroxypropiophenone (2-hydroxy-1-phenyl-propane-1-one); PAC, phenylacetylcarbinol (1-hydroxy-1-phenyl-propane-2-one); PDC, pyruvate decarboxylase; PDCZ.m., pyruvate decarboxylase from Zymomonas mobilis, PDCS.c., pyruvate decarboxylase from Saccharomyces cerevisiae; ThDP, thiamin diphosphate; wt, wild-type.
- Issue published online: 25 DEC 2001
- Article first published online: 25 DEC 2001
- (Received 18 May/6 August 1998)
- Cited By
- 2-hydroxy ketone;
- pyruvate decarboxylase;
- site-directed mutagenesis;
- thiamin diphosphate;
The homotetrameric pyruvate decarboxylase (PDC) from Zymomonas mobilis requires the cofactors thiamin diphosphate and Mg2+ for catalytic activity. We have investigated the role of various amino acid residues in the direct environment of the active site. The role of residue E473 in the catalytic activity and stability of the enzyme was probed by several mutations. All mutant enzymes were either inactive or failed to give any recombinant protein. The close interaction of E473 and N482, which can be deduced from the X-ray structure, has been probed by mutagenesis of N482 to D. This mutation has a significant influence especially on the carboligation reaction of PDC, whereas the binding of the cofactors and the thermostability were not affected. These data suggest a specific interaction of N482 and E473 which is essential for coordinating the second aldehyde molecule during carboligation.
Three hydrophobic residues (L112, I472 and I476) in the vicinity of the active centre have been investigated with respect to their potential influence on the transition states during catalysis. In contrast to L112, I472 and I476 influence the decarboxylation and carboligation reactions. The enlarged substrate-binding site of PDCI472A allows the decarboxylation of longer aliphatic 2-keto acids (C4-C6) as well as aromatic 2-keto acids besides pyruvate. Carboligations using PDCI472A as a catalyst yielded 2-hydroxypropiophenone, benzoin and phenylacetylcarbinol. The enantioselectivity of PAC formation is impaired by mutations of both I472 and I476. The stereochemistry is most significantly affected with the mutant enzyme PDCI476E, which catalyses predominantly the synthesis of (S)-phenylacetylcarbinol.