THE NUCLEOSIDE DIPHOSPHATE KINASE OF MYCOBACTERIUM-SMEGMATIS - IDENTIFICATION OF PROTEINS THAT MODULATE SPECIFICITY OF NUCLEOSIDE TRIPHOSPHATE SYNTHESIS BY THE ENZYME

We report the purification and characterization of the enzyme nucleosi de diphosphate kinase (Ndk) from Mycobacterium smegmatis. The N-termin us of the enzyme was blocked but an internal sequence showed approx. 7 0% homology with the same enzymes from Pseudomonas aeruginosa and Esch erichia coil. Immobilization of the mycobacterial nucleoside diphospha te kinase on a Sepharose 4B matrix and passing the total cell extract through it revealed four proteins (P-70, P-65, P-60, and P-50, respect ively) of M-r 70 kDa, 65 kDa, 60 kDa and 50 kDa that were retained by the column. While the proteins of M-r 70 kDa and 50 kDa modulated the activity of Ndk directing it towards GTP synthesis, the 60 kDa protein channelled the specificity of Ndk entirely towards CTP synthesis. The 65 kDa protein modulated the specificity of Ndk directing it entirely towards UTP synthesis. The specificity for such mycobacterial protein s towards NTP synthesis is retained when they are complexed with P. ae ruginosa Nbk. We further demonstrate that the P-70 protein is pyruvate kinase and that each of the four proteins forms a complex with Ndk an d alters its substrate specificity. Given the ubiquitous nature of Ndk in the living cell and its role in maintaining correct ratios of intr acellular nucleoside triphosphates, the implications of the occurrence of these complexes have been discussed in relation to the precursor p ool for cell wall biosynthesis as well as RNA/DNA synthesis.