The class C acid phosphatase of Helicobacter pylori is a 5' nucleotidase.
The results from purification and characterization studies of the hppA gene product of Helicobacter pylori confirm its identification as a class C acid phosphatase. The hppA gene of H. pylori ATCC strain 49503 was amplified and modified by PCR, cloned into pET21b, and overexpressed in Escherichia coli. The recombinant protein was liberated from membranes and purified (16x) to apparent homogeneity with cation exchange and Ni-chelate chromatography resulting in a recovery of 39% of total starting activity. The recombinant acid phosphatase exhibited a denatured molecular mass of 24 kDa by SDS-PAGE. Phosphatase activity in both crude and purified samples could be renatured and detected after SDS-PAGE. The native molecular mass of recombinant enzyme was approximately 72 kDa by gel filtration chromatography on Superdex 75. While phosphate and tartrate had little effect on phosphatase activity, molybdate, vanadate, and EDTA had significant inhibitory effects on enzymatic activity. Phosphomonoesterase activity for hydrolysis of p-nitrophenylphosphate (pNPP) as well as other substrates was enhanced in the presence of divalent cations including Cu(2+), Ni(2+), Co(2+), and Mg(2+). Recombinant HppA had narrow substrate specificity with highest activity for arylphosphates and significant activity for 5' nucleoside monophosphates. The pH optimum for enzyme activity was 4.6 and 5.2 for purine and pyrimidine 5' monophosphates, respectively. The affinity constants for the 5' nucleoside monophosphates were found to be 0.5-1 mM. Results from this study confirm HppA inclusion in the class C acid phosphatases and led to its identification as a 5' nucleotidase.
Reilly
Thomas J
TJ
Veterinary Medicine Diagnostic Laboratory, E117 Veterinary Medical Sciences Bldg., College of Veterinary Medicine, University of Missouri, Columbia, MO 65211, USA. reillyt@missouri.edu
Calcutt
Michael J
MJ
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012265:Ribonucleotides; D004492:Edetic Acid; D000135:Acid Phosphatase; C465356:HppA protein, Helicobacter pylori; D010744:Phosphoric Monoester Hydrolases; D015720:5'-Nucleotidase