ACTIVE-SITE MUTANTS OF PYRUVATE DECARBOXYLASE FROM ZYMOMONAS-MOBILIS - A SITE-DIRECTED MUTAGENESIS STUDY OF L112, I472, I476, E473 AND N482

The homotetrameric pyruvate decarboxylase (PDC) from Zymomonas mobilis requires the cofactors thiamin diphosphate and Mg2+ for catalytic act ivity. We have investigated the role of various amino acid residues in the direct environment of the active site. The role of residue E473 i n the catalytic activity and stability of the enzyme was probed by sev eral mutations. All mutant enzymes were either inactive or failed to g ive any recombinant protein. The close interaction of E473 and N482, w hich can be deduced from the X-ray structure, has been probed by mutag enesis of N482 to D. This mutation has a significant influence especia lly on the carboligation reaction of PDC, whereas the binding of the c ofactors and the thermostability were not affected. These data, sugges t a specific interaction of N482 and E473 which is essential for coord inating the second aldehyde molecule during carboligation. Three hydro phobic residues (L112, I472 and I476) in the vicinity of the active ce ntre have been investigated with respect to their potential influence on the transition states during catalysis. Tn contrast to L112, I472 a nd I476 influence the decarboxylation and carboligation reactions. The enlarged substrate-binding site of PDCI472A allows the decarboxylatio n of longer aliphatic 2-keto acids (G(4)-C-6) as well as aromatic 2-ke to 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 synthes is of (S)-phenylacetylcarbinol.