Replacement of arginine-171 and aspartate-453 in Streptomyces coelicolor malate synthase A by site-directed mutagenesis inactivates the enzyme

Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the conden sation of glyoxylate and acetyl-CoA to yield malate and CoA. Escherichia co li is known to possess two forms of malate synthase, A and G respectively. The recent elucidation of the E. coli malate synthase G crystal structure s uggested two residues, Arg338 and Asp631, are essential for catalysis. Mult iple sequence alignment of 26 known malate synthase enzymes revealed that t he two proposed sites are highly conserved, despite the low homologies betw een the two distinct forms of the enzyme (13-18%). The conservation of thes e residues in both forms of malate synthase suggests that they possess a si milar catalytic strategy. Thus, despite the absence of a three-dimensional structure for malate synthase A, the significance of this enzyme in the pri mary metabolic pathway has prompted the investigation of the involvement of the corresponding residues, Arg171 and Asp453, in Streptomyces coelicolor malate synthase A by site-directed mutagenesis. Heterologous expression in E. coli followed by purification of the constructed mutant proteins, Arg171 Leu and Asp453Ala, were performed and subsequent enzyme assays of the purif ied mutant proteins indicated a significant loss of catalytic activity, thu s attesting to the need for the corresponding conserved residues to maintai n malate synthase functionality.