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 condensation of glyoxylate and acetyl-CoA to yield malate and CoA. Escherichia coli 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 suggested two residues, Arg338 and Asp631, are essential for catalysis. Multiple sequence alignment of 26 known malate synthase enzymes revealed that the two proposed sites are highly conserved, despite the low homologies between the two distinct forms of the enzyme (13-18%). The conservation of these residues in both forms of malate synthase suggests that they possess a similar catalytic strategy. Thus, despite the absence of a three-dimensional structure for malate synthase A, the significance of this enzyme in the primary 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, Arg171Leu and Asp453Ala, were performed and subsequent enzyme assays of the purified mutant proteins indicated a significant loss of catalytic activity, thus attesting to the need for the corresponding conserved residues to maintain malate synthase functionality.
Goh
L L
LL
Department of Microbiology, Faculty of Medicine, National University of Singapore, Singapore.
Loke
P
P
Sim
T S
TS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008292:Malate Synthase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D001224:Aspartic Acid; D001120:Arginine; D008292:Malate Synthase