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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
Author(s) -
Jung JinWon,
An Jae Hyung,
Na Kyu Bong,
Kim Yu Sam,
Lee Weontae
Publication year - 2000
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.9.7.1294
Subject(s) - active site , mutagenesis , chemistry , site directed mutagenesis , stereochemistry , nuclear magnetic resonance spectroscopy , substrate (aquarium) , nuclear overhauser effect , binding site , malonate , enzyme , two dimensional nuclear magnetic resonance spectroscopy , biochemistry , biology , mutation , ecology , mutant , gene
Abstract 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.