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.