SEQUENCE OF THE BPHD GENE ENCODING 2-HYDROXY-6-OXO-(PHENYL/CHLOROPHENYL)HEXA-2,4-DIENOIC ACID (HOP/CPDA) HYDROLASE INVOLVED IN THE BIPHENYL POLYCHLORINATED BIPHENYL DEGRADATION PATHWAY IN COMAMONAS-TESTOSTERONI - EVIDENCE SUGGESTING INVOLVEMENT OF SER(112) IN CATALYTIC ACTIVITY

The nucleotide sequence of bphD, encoding 2-hydroxy-6-oxo-(phenyl/chlorophenyl) hexa-2,4-dienoic acid hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway of Comamonas testosteroni strain B-356, was determined. Comparison of the deduced amino-acid sequence with published sequences led to the identification of a 'lipase box', containing a consensus pentapeptide sequence GlyXaaSerXaaGly. This suggested that the mechanism of action of this enzyme may involve an Asp-Ser-His catalytic triad similar to that of classical lipases and serine hydrolases. Further biochemical and genetic evidence for the active-site involvement of Ser(112) was obtained by showing that a semipurified enzyme was inhibited by PMSF, a classic inhibitor of serine hydrolases, and by site-directed Ser(112)-->Ala mutagenesis.