Identification of active-site residues in Bradyrhizobium japonicum malonyl-coenzyme A synthetase

Malonyl-CoA synthetase (MCS) has been previously purified and characterized from Bradyrhizobium japonicum USDA 110, The gene encoding this enzyme is n ow cloned, sequenced, and expressed in Escherichia coli. The enzyme contain s 509 amino acid residues, with a calculated molecular mass of 55,239 Da. T he recombinant enzyme was also purified from the transformed E. coli. The e nzyme was essentially indistinguishable from the MCS of B. japonicum by the criteria of polyacrylamide gel electrophoresis and biochemical properties. Based on inhibitor studies of Rhizobium trifolii MCS reported previously a nd database analysis, Arg173, Lys175, His211, and Glu308 were selected for site-directed mutagenesis in order to identify amino acid residues essentia l for substrate binding and/or catalysis. Five different mutant enzymes (R1 73G, K175M, H211L, K175M/H211L, and E308Q) were prepared and then subjected to steady-state kinetic studies, The kinetic data measured for the mutants suggest that Lys175 and His211 participate in the formation of malonyl-AMP , whereas Glu308 may play a role in malonate binding. (C) 2000 Academic Pre ss.