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

• Published in: Protein science 2000-07, Vol.9 (7), p.1294-1303
• Main Authors: JUNG, JIN-WON, AN, JAE HYUNG, NA, KYU BONG, KIM, YU SAM, LEE, WEONTAE
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; adenylate‐forming enzyme; Amino Acid Sequence; Bacterial Proteins; Binding Sites; Coenzyme A - metabolism; Coenzyme A Ligases - chemistry; Coenzyme A Ligases - genetics; Coenzyme A Ligases - metabolism; Magnetic Resonance Spectroscopy - methods; Malonates - metabolism; malonyl‐CoA; malonyl‐CoA synthetase; Models, Molecular; molecular modeling; Molecular Sequence Data; Mutagenesis, Site-Directed; NMR; Protein Conformation
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.9.7.1294

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.