IDENTIFICATION, CHARACTERIZATION, AND CLONING OF A PHOSPHONATE MONOESTER HYDROLASE FROM BURKHOLDERIA-CARYOPHILLI PG2982

The glyphosate degrading bacterium, Burkholderia caryophilli PG2982, w as observed to utilize glyceryl glyphosate as a sole phosphorus source . The hydrolysis of glyceryl glyphosate to glyphosate by a phosphonate ester hydrolase (PEH) was identified as the first metabolic step in t he mineralization pathway, This observation provides the first biologi cal role for a phosphonate ester hydrolase activity, Purified PEH enzy me hydrolyzed several phosphonate esters including p-nitrophenyl pheny lphosphonate, beta-naphthyl phenylphosphonate, and 5-bromo-4-chloro-3- indolyl phenylphosphonate, The purified PEH also hydrolyzed some phosp hodiesters including p-nitrophenyl 5'-thymidine monophosphate and p-ni trophenyl phosphorylcholine. The most catalytically efficient substrat e identified was bis-(p-nitrophenyl) phosphate with a K-m of 0.9 mM an d a k(cat) of 6.2 x 10(2) min(-1), suggesting that the enzyme may also function in vice as a phosphodiesterase. The native enzyme was a homo tetramer of 58-kDa subunits and exhibited a pi of 4.2. The enzyme acti vity had a pH activity optimum of 9.0 and was stimulated 14-fold by Mn 2+ ions, but a metal cofactor was not essential for activity, N-termin al and tryptic fragment amino acid sequences were obtained from the pu rified PEH protein and used to clone the B. caryophilli PG2982 gene, d esignated pehA. The unique substrate specificity of the enzyme and pot ential use as a novel conditional lethal gene in plants are discussed.