The role of residues glutamate-50 and phenylalanine-496 in Zymomonas mobilis pyruvate decarboxylase

Abstract

Several enzymes require thiamine diphosphate (ThDP) as an essential cofactor, 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, homotetrameric subunit structure and simple kinetic properties. Crystallographic analyses of three ThDP-dependent enzymes [Müller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103] have suggested that an invariant glutamate participates in catalysis. In order to evaluate the role of this residue, identified in PDC from Zymomonas mobilis as Glu-50, it has been altered to glutamine and aspartate by site-directed mutagenesis of the cloned gene. The mutant proteins were expressed in Escherichia coli. Here we demonstrate that substitution with aspartate yields an enzyme with 3% of the activity of the wild-type, but with normal kinetics for pyruvate. Replacement of Glu-50 with glutamine yields an enzyme with only 0.5% of the catalytic activity of the wild-type enzyme. Each of these mutant enzymes has a decreased affinity for both ThDP and Mg2+. It has been reported that the binding of cofactors to apoPDC quenches the intrinsic tryptophan fluorescence [Diefenbach and Duggleby (1991) Biochem. J. 276, 439-445] and we have identified the residue responsible as Trp-487 [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98]. Although this residue is some distance from the cofactor binding site, it lies in the dimer interface, and the proposal has been put forward [Dyda, Furey, Swaminathan, Sax, Farrenkopf and Jordan (1993) Biochemistry 32, 6165-6170] that alteration of ring stacking with Phe-496 of the adjacent subunit is the mechanism of fluorescence quenching when cofactors bind. The closely related enzyme indolepyruvate decarboxylase (from Enterobacter cloacae) has a leucine residue at the position corresponding to Phe-496 but shows fluorescence quenching properties that are similar to those of PDC. This suggests that the fluorescence quenching is due to some perturbation of the local environment of Trp-487 rather than to a specific interaction with Phe-496. This latter hypothesis is supported by our data: mutation of this phenylalanine to leucine, isoleucine or histidine in PDC does not eliminate the fluorescence quenching upon addition of cofactors.

Covalently Bound Substrate at the Regulatory Site of Yeast Pyruvate Decarboxylases Triggers Allosteric Enzyme Activation

“…His-114 and His-115, the next upstream neighbors of loop 104 -113, are part of the active site. For His-114, an essential function in PDC catalysis has been proposed from kinetic studies with accordant variants from yeast and bacteria (7,8,42,43). Tittmann et al (44) postulated a specific role for His-114 (together with Asp-28) during release of the reaction product acetaldehyde.…”

Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase

“…expression vectors [30] with which we have had some success in high-level expression of pyruvate decarboxylase [31]. Putting the coding sequence into this vector tripled the expression.…”

The Role of His113 and His114 in Pyruvate Decarboxylase from Zymomonas Mobilis

“…Robinson and Chun (1993) came to similar conclusions using a sequence alignment of PDC, transketolase, and the El component of the pyruvate dehydrogenase complexes of Homo sapiens, Bacillus stearotherrnuphilus and Escherichia coli. However, this alignment failed to identify the conserved Glu residues that are now known to be required for catalysis in transketolase (Wikner et al, 1994) and PDC (Candy et al, 1996a). While the crystal structures Arjunan et al, 1996) and mutagenesis studies (Diefenbach et al, 1992;Candy and Duggleby, 1994;Candy et al, 1996a) of PDC have shed light on the binding of the cofactors and the reaction mechanism, the interaction between substrate and enzyme remains unclear.…”

“…However, this alignment failed to identify the conserved Glu residues that are now known to be required for catalysis in transketolase (Wikner et al, 1994) and PDC (Candy et al, 1996a). While the crystal structures Arjunan et al, 1996) and mutagenesis studies (Diefenbach et al, 1992;Candy and Duggleby, 1994;Candy et al, 1996a) of PDC have shed light on the binding of the cofactors and the reaction mechanism, the interaction between substrate and enzyme remains unclear. In contrast, the three-dimensional structure of yeast transketolase shows an identifiable substrate channel located at the subunit interface.…”