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Volume 271, Number 42, Issue of October 18, 1996 pp. 25754-25761
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Identification, Characterization, and Cloning of a Phosphonate Monoester Hydrolase from Burkholderia caryophilli PG2982

(Received for publication, June 19, 1996, and in revised form, July 23, 1996)

Stanton B. Dotson , Christine E. Smith ^¶ , Cliff S. Ling , Gerard F. Barry ^'' and Ganesh M. Kishore ^''

From the  Glycobiology Group, G. D. Searle, St. Louis, Missouri 63167, the ^¶ Protein Biochemistry Group, G. D. Searle, Chesterfield, Missouri 63198, the  Analytical Sciences Center, Monsanto Corporate Research, Saint Louis, Missouri 63167, and ^'' Ceregen, A Unit of Monsanto Company, Chesterfield, Missouri 63198

The glyphosate-degrading bacterium, Burkholderia caryophilli PG2982, was 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 the mineralization pathway. This observation provides the first biological role for a phosphonate ester hydrolase activity. Purified PEH enzyme hydrolyzed several phosphonate esters including p-nitrophenyl phenylphosphonate, -naphthyl phenylphosphonate, and 5-bromo-4-chloro-3-indolyl phenylphosphonate. The purified PEH also hydrolyzed some phosphodiesters including p-nitrophenyl 5-thymidine monophosphate and p-nitrophenyl phosphorylcholine. The most catalytically efficient substrate identified was bis-(p-nitrophenyl) phosphate with a K_m of 0.9 mM and a k_cat of 6.2 × 10² min¹, suggesting that the enzyme may also function in vivo as a phosphodiesterase. The native enzyme was a homotetramer of 58-kDa subunits and exhibited a pI of 4.2. The enzyme activity had a pH activity optimum of 9.0 and was stimulated 14-fold by Mn²⁺ ions, but a metal cofactor was not essential for activity. N-terminal and tryptic fragment amino acid sequences were obtained from the purified PEH protein and used to clone the B. caryophilli PG2982 gene, designated pehA. The unique substrate specificity of the enzyme and potential use as a novel conditional lethal gene in plants are discussed.

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