Escherichia coli cyclopropane fatty acid synthase.
Escherichia coli fatty acid cyclopropane synthase (CFAS) was overproduced and purified as a His6-tagged protein. This recombinant enzyme is as active as the native enzyme with a Km of 90 microm for S-AdoMet and a specific activity of 5 x 10(-2) micromol.min(-1).mg(-1). The enzyme is devoid of organic or metal cofactors and is unable to catalyze the wash-out of the methyl protons of S-AdoMet to the solvent, data that do not support the ylide mechanism. Inactivation of the enzyme by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a pseudo first-order process with a rate constant of 1.2 m(-1).s(-1), is not protected by substrates. Graphical analysis of the inactivation by DTNB revealed that only one cysteine is responsible for the inactivation of the enzyme. The three strictly conserved Cys residues among cyclopropane synthases, C139, C176 and C354 of the E. coli enzyme, were mutated to serine. The relative catalytic efficiency of the mutants were 16% for C139S, 150% for C176S and 63% for C354S. The three mutants were inactivated by DTNB at a rate comparable to the rate of inactivation of the His6-tagged wild-type enzyme, indicating that the Cys responsible for the loss of activity is not one of the conserved residues. Therefore, none of the conserved Cys residues is essential for catalysis and cannot be involved in covalent catalysis or general base catalysis. The inactivation is probably the result of steric hindrance, a phenomenon irrelevant to catalysis. It is very likely that E. coli CFAS operates via a carbocation mechanism, but the base and nucleophile remain to be identified.
Courtois
Fabienne
F
Laboratoire de Chimie Organique Biologique, UMR7613 CNRS, Université Pierre et Marie Curie, Paris, France.
Guérard
Christine
C
Thomas
Xavier
X
Ploux
Olivier
O
D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed
D017931:DNA Primers; D004791:Enzyme Inhibitors; D004228:Dithionitrobenzoic Acid; D008780:Methyltransferases; C019964:cyclopropane synthetase