Cloning, functional expression, and complementation analysis of an inorganic pyrophosphatase from Bartonella bacilliformis.
We have cloned the inorganic pyrophosphatase gene (ppa) from the facultative intracellular pathogen Bartonella bacilliformis and characterized its encoded product. The 531-bp gene is located approximately 1 kb downstream of, and in opposite orientation to, the invasion-associated locus (ialAB) of B. bacilliformis. The predicted protein encoded by ppa is 177 amino acid residues, which is in agreement with in vitro and in vivo synthesis of a protein with an apparent molecular mass of 22-23 kDa. The predicted B. bacilliformis pyrophosphatase (PPase) sequence is 53% identical and 85% similar to the E. coli PPase (EC 3.6.1.1), and contains all 12 of the amino acid residues implicated in the catalytic active site. The isolated B. bacilliformis PPase exhibits an activity of 51 +/- 2 mumol PO4 released/(mg protein.min) at 28 degrees C and pH 8, and is sensitive to inhibition by Ca2+. In keeping with other prokaryotic PPases, B. bacilliformis PPase activity occurs from pH 6 to 10 (optimal pH = 8) and demonstrates high thermostability in the presence of Mg2+ (highest activity at 55 degrees C, relative activity = 80 +/- 3% at pH 8). The cloned B. bacilliformis ppa is able to genetically complement a ppa- mutant strain of E. coli.
Mitchell
S J
SJ
Division of Biological Sciences, University of Montana, Missoula 59812-1002, USA.
Minnick
M F
MF
D000595:Amino Acid Sequence; D001473:Bartonella / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D043564:Inorganic Pyrophosphatase; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011755:Pyrophosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D002118:Calcium