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 now cloned, sequenced, and expressed in Escherichia coli. The enzyme contains 509 amino acid residues, with a calculated molecular mass of 55,239 Da. The recombinant enzyme was also purified from the transformed E. coli. The enzyme 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 and database analysis, Arg173, Lys175, His211, and Glu308 were selected for site-directed mutagenesis in order to identify amino acid residues essential for substrate binding and/or catalysis. Five different mutant enzymes (R173G, 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.
Koo
H M
HM
Department of Biochemistry, College of Science, Bioproducts Research Center, Yonsei University, Seoul, Korea.
Kim
Y S
YS
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D020369:Bradyrhizobium / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D003066:Coenzyme A Ligases; C021533:malonyl-CoA synthetase