Characterization of a phosphomonoesterase from Brucella abortus.
Brucellae are facultative intracellular bacterial pathogens that reside primarily in cells of the reticuloendothelial system. The high-speed supernatant obtained after centrifuging a suspension of Brucella abortus that had been frozen-thawed and sonicated contained abundant phosphomonoesterase activity, determined by using 4-methylumbelliferylphosphate as the substrate; this enzyme was purified 2,900-fold (yield, 570%) by chromatography on DE-52 cellulose and hydroxylapatite columns and high-performance liquid chromatography-gel filtration. The native enzyme had a molecular mass of 120,000 daltons (+/- 10,000 daltons), as determined by gel filtration chromatography, and resolved into two bands (60,000 and 66,000 daltons) when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The B. abortus phosphomonoesterase had the following properties: pH optimum, 6.0 to 6.5; isoelectric point, 3.0; substrate specificity, 5'-AMP greater than 3'-AMP greater than 3'-GMP greater than 5'-GDP greater than 5'-CDP greater than 5'-CTP greater than 5'-UPT greater than phosphotyrosine greater than phosphoserine greater than phosphothreonine. The Km for 5'-AMP was 0.37 mM. Phosphatidylinositol 4,5-bisphosphate and myo-inositol 1,3,4-trisphosphate were poor substrates for the B. abortus enzyme. The phosphomonoesterase did not inhibit superoxide anion production by human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine. The phosphomonoesterase may be one of the bacterial enzymes in the pathway leading to the production of adenine, which is secreted by B. abortus and blocks the activation of neutrophils.
Saha
A K
AK
Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Mukhopadhyay
N K
NK
Dowling
J N
JN
Ficht
T A
TA
Adams
L G
LG
Glew
R H
RH
D000135:Acid Phosphatase / Q000302:isolation & purification; D002003:Brucella abortus / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D009504:Neutrophils / Q000502:physiology; D009708:Nucleotidases / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity; D013481:Superoxides / Q000378:metabolism
D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D013481:Superoxides; D015544:Inositol 1,4,5-Trisphosphate; D009708:Nucleotidases; D000135:Acid Phosphatase; D010744:Phosphoric Monoester Hydrolases; D006026:Glycoside Hydrolases