Mutagenesis at asp27 of pyruvate decarboxylase from Zymomonas mobilis. Effect on its ability to form acetoin and acetolactate.
Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a bivalent cation as essential cofactors. The three-dimensional structure of PDC from Zymomonas mobilis (ZMPDC) shows that Asp27 (D27) is close to ThDP in the active site, and mutagenesis of this residue has suggested that it participates in catalysis. The normal product of the PDC reaction is acetaldehyde but it is known that the enzyme can also form acetoin as a by-product from the hydroxyethyl-ThDP reaction intermediate. This study focuses on the role of D27 in the production of acetoin and a second by-product, acetolactate. D27 in ZMPDC was altered to alanine (D27A) and this mutated protein, the wild-type, and two other previously constructed PDC mutants (D27E and D27N) were expressed and purified. Determination of the kinetic properties of D27A showed that the affinity of D27A for ThDP is decreased 30-fold, while the affinity for Mg2+ and the Michaelis constant for pyruvate were similar to those of the wild-type. The time-courses of their reactions were investigated. Each mutant has greatly reduced ability to produce acetaldehyde and acetoin compared with the wild-type PDC. However, the effect of these mutations on acetaldehyde production is greater than that on acetoin formation. The D27A mutant can also form acetolactate, whereas neither of the other mutants, nor the wild-type PDC, can do so. In addition, acetaldehyde formation and/or release are reversible in wild-type ZMPDC but irreversible for the mutants. The results are explained by a mechanism involving thermodynamic and geometric characteristics of the intermediates in the reaction.
Wu
Y G
YG
Life Science College of Jilin University, Changchun, China; Centre for Protein Structure, Function and Engineering, Department of Biochemistry, University of Queensland, Brisbane, Queensland, Australia.
Chang
A K
AK
Nixon
P F
PF
Li
W
W
Duggleby
R G
RG
D000079:Acetaldehyde / Q000494:pharmacology; D000093:Acetoin / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D007700:Kinetics; D007773:Lactates / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics
D007773:Lactates; D011994:Recombinant Proteins; D001224:Aspartic Acid; C006359:alpha-acetolactate; D019289:Pyruvic Acid; D000093:Acetoin; D011767:Pyruvate Decarboxylase; D000079:Acetaldehyde