The active site and substrates binding mode of malonyl-CoA synthetase determined by transferred nuclear Overhauser effect spectroscopy, site-directed mutagenesis, and comparative modeling studies.
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 alpha-helices, 24 beta-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.
Jung
J W
JW
Department of Biochemistry, College of Science, Yonsei University, Shinchon-Dong, Seoul, Korea.
An
J H
JH
Na
K B
KB
Kim
Y S
YS
Lee
W
W
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D003065:Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008314:Malonates / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D001426:Bacterial Proteins; D008314:Malonates; D000255:Adenosine Triphosphate; C030290:malonic acid; D003066:Coenzyme A Ligases; C021533:malonyl-CoA synthetase; D003065:Coenzyme A