The active sites and substrate bindings of Rhizobium
trifolii molonyl-CoA synthetase (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 consists of 16 α-helices,
24 β-strands, and several long loops. The core active
site was determined as a wide cleft close to the end of
the small C-terminal domain. The catalytic substrate malonate
is placed between ATP and His206 in the MCS enzyme, supporting
His206 in its catalytic role as it generates reaction intermediate,
malonyl-AMP. These findings are strongly supported by previous
biochemical data, as well as by the site-directed mutagenesis
data reported here. This structure reveals the biochemical
role as well as the substrate specificity that conservative
residues of adenylate-forming enzymes have.