Identification, characterization, and cloning of a phosphonate monoester hydrolase from Burkholderia caryophilli PG2982.
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, beta-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 Km of 0.9 mM and a kcat of 6.2 x 10(2) min-1, 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.
Dotson
S B
SB
Glycobiology Group, G. D. Searle, St. Louis, Missouri 63167, USA.
Smith
C E
CE
Ling
C S
CS
Barry
G F
GF
Kishore
G M
GM
D000595:Amino Acid Sequence; D000838:Anions / Q000494:pharmacology; D001483:Base Sequence; D019117:Burkholderia / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D004950:Esterases / Q000737:chemistry* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005990:Glycerol / Q000031:analogs & derivatives / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D006540:Herbicides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D019216:Metals, Heavy / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D013379:Substrate Specificity; D000097797:Glyphosate
D000838:Anions; D004950:Esterases; D005990:Glycerol; D005998:Glycine; D006540:Herbicides; D019216:Metals, Heavy; D000097797:Glyphosate; C101868:glyceryl glyphosate; C101869:phosphonate monoester hydrolase