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 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 EA73 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.
Pohl
M
M
Institut für Enzymtechnologie der Heinrich-Heine Universität Düsseldorf, Forschungszentrum Jülich, Germany.
Siegert
P
P
Mesch
K
K
Bruhn
H
H
Grötzinger
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013835:Thiamine Pyrophosphate / Q000378:metabolism; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate