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Stereospecificity of 2-Monochloropropionate Dehalogenation by the Two Dehalogenases of Pseudomonas putida PP 3 : Evidence for Two Different Dehalogenation Mechanisms

Pseudomonas putida PP3 grew on DL-2-monochloropropionate (2MC PA) with a release of chloride ions consistent with the dechlorination of both isomers. The organism grew on either D-or L-2MCPA. Dehalogenase activity in cell-free extracts showed that both D-and L-~ M C PA were dehalogenated. Pseudomonas putida PP3 contains two dehalogenases, and studies with the separated enzymes revealed that the fraction I enzyme used both D-and L-2MCPA, the rate of dechlorination of L-2MCPA being 80% of the rate of D-2MCPA dechlorination. The product of the reaction, lactate, retained the same optical configuration as the substrate provided. The fraction I1 dehalogenase also dechlorinated D-and L-2MCPA, with the same difference in rates as for the fraction I dehalogenase, but the lactates produced were of the opposite configuration to their precursors. The two dehalogenases showed further differences with respect to inhibition by two sulphydryl-blocking agents, N-ethylmaleimide and p-chloromercuribenzoate. Fraction I dehalogenase was considerably more sensitive to these two reagents compared with the fraction I1 dehalogenase. Dithiothreitol partially protected the fraction I dehalogenase from N-ethylmaleimide inhibition. The results are discussed in terms of the possible evolutionary relationships of the two dehalogenases. Several studies have indicated that bacteria contain a number of different dehalogenases particularly with respect to their substrate specificities, electrophoretic mobilities and inhibition by sulphydryl-blocking agents a, b). It has been found that Pseudomonas putida PP3, isolated by Senior et al. (1976) from a microbial community growing on 22DCPA, contains two dehalogenases with different substrate specificities and thermal stabilities (Weightman et al., 1979). None of these previous studies sought to resolve the relationship, or otherwise, between these different enzymes. It was suggested that the fraction I and I1 dehalogenases of P. putida PP3 may have evolved from a common ancestral enzyme (Weightman et al., 1979), a conclusion tentatively drawn from the two enzymes' substrate specificities. The aim of the present work was to examine those properties of the two P. putida PP3 enzymes which might indicate whether or not the enzymes were mechanistically related. This was examined by investigating their dehalogenation of the optically active isomers of 2MCPA and their response towards sulphydryl-blocking agents.