Two forms of the nucleoside diphosphate kinase of Pseudomonas aeruginosa 8830: altered specificity of nucleoside triphosphate synthesis by the cell membrane-associated form of the truncated enzyme.
Nucleoside diphosphate kinase (EC 2.7.4.6) (Ndk) is a ubiquitous enzyme functioning in the intracellular distribution of terminal phosphate bond energy among the various nucleotides used in synthetic and regulatory functions in cells. We have previously reported that in Pseudomonas aeruginosa, this important enzyme is transcriptionally regulated by the gene algR2 and posttranslationally regulated by a phosphoprotein phosphatase for the phosphorylated form of Ndk. We report here that an intracellular protease cleaves the 16-kDa form of Ndk to a 12-kDa form that undergoes autophosphorylation with an efficiency almost identical to that of the 16-kDa form. The 12-kDa form was found to be predominantly associated with the P. aeruginosa cell membrane fraction, whereas the 16-kDa form was predominantly cytoplasmic. In the membrane-associated state, the 12-kDa form of Ndk was found to synthesize GTP in preference to other nucleoside triphosphates. The specificity toward GTP synthesis could be abolished by the addition of Tween 20 or Triton X-100. The activity itself could be abolished by the addition of anti-Ndk antibody to the assay mixture. The formation of the 12-kDa form of Ndk and its association with the cell membrane were found to be related to the growth stage of P. aeruginosa, with less than 1% of the 12-kDa Ndk detectable in the membrane fraction at early log phase in comparison with the levels present at late stationary phase.
Shankar
S
S
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago , Illinois 60612, USA.
Kamath
S
S
Chakrabarty
A M
AM
D003593:Cytoplasm / Q000378:metabolism; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000378:metabolism*; D009711:Nucleotides / Q000096:biosynthesis*; D010766:Phosphorylation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity
D008565:Membrane Proteins; D009711:Nucleotides; D009701:Nucleoside-Diphosphate Kinase; D010450:Endopeptidases