Identification of residues essential for a two-step reaction by malonyl-CoA synthetase from Rhizobium trifolii

Malonyl-CoA synthetase (MCS) catalyses the formation of malonyl-CoA in a tw o-step reaction consisting of the adenylation of malonate with ATP followed by malonyl transfer from malonyl-AMP to CoA. In order to identify amino ac id residues essential for each step of the enzyme, catalysis based on chemi cal modification and database analysis, Arg-168, Lys-170, and His-206 were selected for site-directed mutagenesis. Glutathione-S-transferase-fused enz yme (GST-MCS) was constructed and mutagenized to make R168G, K170M, R168G/K 170M and H206L mutants, respectively. The MCS activity of soluble form GST- MCS was the same as that of wild-type MCS. Circular dichroism spectra for t he four mutant enzymes were nearly identical to that for the GST-MCS, indic ating that Arg-168, Lys-170 and His-206 are not important for conformation but presumably for substrate binding and/or catalysis. HPLC analysis of pro ducts revealed that the intermediate malonyl-AMP is not accumulated during MCS catalysis and that none of the mutant enzymes accumulated it either. Kinetic analysis of the mutants revealed that Lys-170 and His-206 play a cr itical role for ATP binding and the formation of malonyl-AMP, whereas Arg-1 68 is critical for formation of malonyl-CoA and specificity for malonyl-AMP . Molecular modelling based on the crystal structures of luciferase and grami cidin S synthetase 1 provided MCS structure which could fully explain all t hese biochemical data even though the MCS model was generated by comparativ e modelling.