Evidence for a gem-Diol Reaction Intermediate in Bacterial C−C Hydrolase Enzymes BphD and MhpC from ¹³C NMR Spectroscopy^†

C−C hydrolase enzymes MhpC and BphD catalyze the hydrolytic C−C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid and Burkholderia xenovorans LB400 biphenyl degradation pathways and are both members of the α/β-hydrolase family containing a Ser-His-Asp catalytic triad. The catalytic mechanism of this family of enzymes is thought to proceed via a gem-diol reaction intermediate, which has not been observed directly. Site-directed single mutants of BphD in which catalytic residues His-265 and Ser-112 were replaced with Ala were found to possess 10⁴-fold reduced k_cat values, and in each case, the C−C cleavage step was shown by pre-steady-state kinetic analysis to be rate-limiting. The processing of a 6-¹³C-labeled aryl-containing substrate by these H265A or S112A mutant BphD enzymes was monitored directly by ¹³C NMR spectroscopy. A new line-broadened signal was observed at 128 ppm for each enzyme, corresponding to the proposed gem-diol reaction intermediate, over a time scale of 1−24 h. A similar signal was observed upon incubation of the ¹³C-labeled substrate with an H114A MhpC mutant, which is able to accept the 6-phenyl-containing substrate, on a shorter time scale. The direct observation of a gem-diol intermediate provides further evidence that supports a general base mechanism for this family of enzymes.