INSIGHTS INTO THE MECHANISM OF CATALYSIS BY THE P-C BOND-CLEAVING ENZYME PHOSPHONOACETALDEHYDE HYDROLASE DERIVED FROM GENE SEQUENCE-ANALYSIS AND MUTAGENESIS

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolys is of phosphonoacetaldehyde to acetaldehyde and inorganic phosphate. I n this study, the genes encoding phosphonatase in Bacillus cereus and in Salmonella typhimurium were cloned for high-level expression in Esc herichia coli. The kinetic properties of the purified, recombinant pho sphonatases were determined. The Schiff base mechanism known to operat e in the B. cereus enzyme was verified for the S. typhimurium enzyme b y phosphonoacetaldehyde-sodium borohydride-induced inactivation and by site-directed mutagenesis of the catalytic lysine 53. The protein seq uence inferred from the B. cereus phosphonatase gene was determined, a nd this sequence was used along with that from the S. typhimurium phos phonatase gene sequence to search the primary sequence databases for p ossible structural homologues. We found that phosphonatase belongs to a novel family of hydrolases which appear to use a highly conserved ac tive site aspartate residue in covalent catalysis. On the basis of thi s finding and the known stereochemical course of phosphonatase-catalyz ed hydrolysis at phosphorus (retention), we propose a mechanism which involves Schiff base formation with lysine 53 followed by phosphoryl t ransfer to aspartate (at position 11 in the S. typhimurium enzyme and position 12 in the B. cereus phosphonatase) and last hydrolysis at the imine C(1) and acyl phosphate phosphorus.