Purification and characterization of YfkN, a trifunctional nucleotide phosphoesterase secreted by Bacillus subtilis.
YfkN isolated from the culture supernatant of Bacillus subtilis in the exponential phase of growth is a protein of 143.5 kDa that derives from a putative large precursor of 159.6 kDa processed at both the N- and C-terminal ends. Pulse-chase experiments indicated that the release occurs slowly with a half-time longer than 30 min, suggesting that the event is coupled with wall turnover. YfkN exhibits 2',3' cyclic nucleotide phosphodiesterase, 2' (or 3') nucleotidase and 5' nucleotidase activities. In vitro the protein is reduced by subtilisin digestion to a shorter polypeptide (68 kDa), displaying phosphodiesterase activity but devoid of any 5'nucleotidase activity. This proteolytic processing led us to localize the potential active sites of the various nucleotidase activities. When bacteria were grown in low phosphate medium, the exocellular production of the enzyme was enhanced, suggesting that it plays a role in phosphate metabolism. Comparison with nucleotidase databases suggests that yfkN resulted from gene fusion.
Chambert
Régis
R
Institut Jacques Monod, CNRS-Universités Paris 6 et Paris 7, Laboratoire Génétique et Membranes, Tour 43, 2, place Jussieu, 75251 Paris, Cedex 05, France. chambert@ccr.jusieu.fr
Pereira
Yannick
Y
Petit-Glatron
Marie-Françoise
MF
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009708:Nucleotidases / Q000302:isolation & purification* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020860:Subtilisin / Q000378:metabolism
D009708:Nucleotidases; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D020860:Subtilisin