INVESTIGATION OF THE COFACTOR-BINDING SITE OF ZYMOMONAS-MOBILIS PYRUVATE DECARBOXYLASE BY SITE-DIRECTED MUTAGENESIS

Several enzymes require thiamin diphosphate (ThDP) as an essential cof actor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. A sequence motif of ap prox. 30 residues, beginning with a glycine-aspartate-glycine (-GDG-) triplet and ending with a double asparagine (-NN-) sequence, has been identified in many of these enzymes [Hawkins, Borges and Perham (1989) FEBS Lett. 255, 77-82]. Other residues within this putative ThDP-bind ing motif are conserved, but to a lesser extent, including a glutamate and a proline residue. The role of the elements of this motif has bee n clarified by the determination of the three-dimensional structure of three of these enzymes [Muller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103]. Four of the residues within thi s motif were modified by site-directed mutagenesis of the cloned PDC g ene to evaluate their role in cofactor binding. The mutant proteins we re expressed in Escherichia coli and found to purify normally, indicat ing that the tertiary structure of these enzymes had not been grossly perturbed by the amino acid substitutions. We have shown previously [D iefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98] t hat changing the aspartate in the -GDG-sequence to glycine, threonine or asparagine yields an inactive enzyme that is unable to bind ThDP, t herefore verifying the role of the ThDP-binding motif. Here we demonst rate that substitution with glutamate yields an active enzyme with a g reatly reduced affinity for both ThDP and Mg2+, but with normal kineti cs for pyruvate. Unlike the wild-type tetrameric enzyme, this mutant p rotein usually exists as a dimer. Replacement of the second asparagine of the -NN- sequence by glutamine also yields an inactive enzyme whic h is unable to bind ThDP, whereas replacement with an aspartate residu e results in an active enzyme with a reduced affinity for ThDP but whi ch displays normal kinetics for both Mg2+ and pyruvate. Replacing the conserved glutamate with aspartate did not alter the properties of the enzyme, while the conserved proline, thought to be required for struc tural reasons, could be substituted with glycine or alanine without in activating the enzyme, but these changes did reduce its stability.