title: Escherichia coli cyclopropane fatty acid synthase: is a bound bicarbonate ion the active-site base?
abstract: Cyclopropane synthases catalyze the cyclopropanation of unsaturated fatty acid using S-adenosyl-L-methionine as the methylene donor. The crystal structure of three cyclopropane synthases from Mycobacterium tuberculosis showed a bicarbonate ion bound in the active site that was proposed to act as a general base in the reaction mechanism [Huang, C., Smith, V., Glickman, M. S., Jacobs, W. R., and Sacchettini, J. C. (2002) J. Biol. Chem. 277, 11559-11569]. Because the in vitro activity of M. tuberculosis cyclopropane synthases has not yet been reported and because the ligands of the bicarbonate ion are all strictly conserved in cyclopropane synthases, we used the closely related Escherichia coli cyclopropane fatty acid synthase for this study. The putative ligands that share a hydrogen bond with the bicarbonate through their side chains were mutated. H266A, Y317F, E239A, and E239Q mutants were thus constructed and purified, and their catalytic efficiencies were 5.3, 0.7, 0.2, and <0.02%, respectively. C139 that is bound to the bicarbonate by its NH amide had already been mutated to serine in a previous work, and this mutant retains 31% of the activity of the wild-type enzyme. Kinetic analyses and binding studies using spectrofluorimetry showed that these mutations affected the catalytic constant rather than the binding of the substrates. While addition of free bicarbonate had almost no effect on the wild-type enzyme activity, all mutants, with the exception of E239A and E239Q, were rescued by the addition of free bicarbonate. The catalytic efficiencies of the rescued mutants were 85, 16, and 14% for C139S, H266A, and Y317F, respectively. This effect was specific to bicarbonate. The kinetic parameters of the rescued mutants were determined, and it is shown that the rescuing effect is due to an increase in kcat. These data are interpreted by assuming that the E. coli cyclopropane fatty acid synthase specifically binds a bicarbonate ion that is involved in catalysis, as proposed for the M. tuberculosis enzymes, and that mutation of the bicarbonate ligands decreases the affinity for that ion. However, because the E239Q mutation could not be rescued, we propose that E239 forms a catalytic dyad with the bicarbonate to perform the proton abstraction necessary in the chemical pathway to the cyclopropane ring.
authors.affiliation: Synthèse Structure et Fonction de Molécules Bioactives, UMR7613 CNRS-UPMC, Université Pierre et Marie Curie, boîte 182, 4, place Jussieu, F-75252 Paris cedex 05, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Courtois
authors.forename: Olivier
authors.initials: O
authors.lastname: Ploux
mesh_terms: D001639:Bicarbonates / Q000737:chemistry*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D013050:Spectrometry, Fluorescence
chemical_list: D001639:Bicarbonates; D008780:Methyltransferases; C019964:cyclopropane synthetase