The active site and substrates binding mode of malonyl-CoA synthetase determined by transferred nuclear Overhauser effect spectroscopy, site-directedmutagenesis, and comparative modeling studies

The active sites and substrate bindings of Rhizobium trifolii molonyl-CoA s ynthetase (MCS) catalyzing the malonyl-CoA formation from malonate and CoA have been determined based on NMR spectroscopy, site-directed mutagenesis, and comparative modeling methods. The MCS-bound conformation of malonyl-CoA was determined from two-dimensional-transferred nuclear Overhauser effect spectroscopy data. MCS protein folds into two structural domains and consis ts of 16 alpha-helices, 24 beta-strands, and several long loops. The core a ctive site was determined as a wide cleft close to the end of the small C-t erminal domain. The catalytic substrate malonate is placed between ATP and His206 in the MCS enzyme, supporting His206 in its catalytic role as it gen erates reaction intermediate, malonyl-AMP. These findings are strongly supp orted by previous biochemical data, as well as by the site-directed mutagen esis data reported here. This structure reveals the biochemical role as wel l as the substrate specificity that conservative residues of adenylate-form ing enzymes have.