Sequence analysis of nutA gene encoding membrane-bound Cl(-)-dependent 5'-nucleotidase of Vibrio parahaemolyticus.
The membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus is unique in requiring Cl- for activity. We cloned the nutA gene encoding the 5'-nucleotidase and sequenced it. It contained an open reading frame consisting of 1,680 nucleotides capable of encoding a protein of 560 amino acid residues. The first 21 amino acid residues of the N-terminal portion of this protein seem to be a signal peptide. The rest of the polypeptide (539 residues) is hydrophilic, and its molecular weight was calculated to be 60,008, which is in good agreement with the value of 63 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the 5'-nucleotidase derived from the cloned nutA gene. We tried to determine the amino acid sequence of the N-terminal portion of the purified enzyme. However, the N-terminal residue seemed to be blocked. As this 5'-nucleotidase can be solubilized from membrane vesicles with detergent, it may be a lipoprotein. The amino acid sequence around the possible cleavage site of the 5'-nucleotidase had homology with the sequences of the cleavage sites of the lipoproteins of Escherichia coli and other bacteria. The amino acid sequence had high (about 60%) homology with the sequence of periplasmic 5'-nucleotidase (uridine diphosphate sugar hydrolase, the product of the ushA gene) of E. coli. It also contained regions that showed some homology with the nucleotide binding sites of many nucleotide binding proteins.
Tamao
Y
Y
Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University.
Noguchi
K
K
Sakai-Tomita
Y
Y
Hama
H
H
Shimamoto
T
T
Kanazawa
H
H
Tsuda
M
M
Tsuchiya
T
T
D015720:5'-Nucleotidase / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D002712:Chlorides / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008566:Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021382:Protein Sorting Signals / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014736:Vibrio parahaemolyticus / Q000201:enzymology* / Q000235:genetics
D002712:Chlorides; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D015720:5'-Nucleotidase