The nucleoside diphosphate kinase of Mycobacterium smegmatis: identification of proteins that modulate specificity of nucleoside triphosphate synthesis by the enzyme

• Published in: Molecular microbiology 1997-05, Vol.24 (3), p.477-487
• Main Authors: Shankar, Sandeep, Hershberger, C. Douglas, Chakrabarty, A. M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Ammonium Sulfate; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Chemical Fractionation; Chromatography, Gel; Enzymes, Immobilized; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Molecular Weight; Mycobacterium - enzymology; Mycobacterium - genetics; Mycobacterium - metabolism; Mycobacterium smegmatis; Nucleoside-Diphosphate Kinase - genetics; Nucleoside-Diphosphate Kinase - isolation & purification; Nucleoside-Diphosphate Kinase - metabolism; Nucleotides - biosynthesis; Pseudomonas aeruginosa; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Sequence Homology, Amino Acid; Species Specificity; Substrate Specificity
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1046/j.1365-2958.1997.3491724.x

We report the purification and characterization of the enzyme nucleoside diphosphate kinase (Ndk) from Mycobacterium smegmatis. The N‐terminus of the enzyme was blocked but an internal sequence showed approx. 70% homology with the same enzymes from Pseudomonas aeruginosa and Escherichia coli. Immobilization of the mycobacterial nucleoside diphosphate kinase on a Sepharose 4B matrix and passing the total cell extract through it revealed four proteins (P70, P65, P60, and P50, respectively) of Mr 70 kDa, 65 kDa, 60 kDa and 50 kDa that were retained by the column. While the proteins of Mr 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 proteins towards NTP synthesis is retained when they are complexed with P. aeruginosa Ndk. We further demonstrate that the P70 protein is pyruvate kinase and that each of the four proteins forms a complex with Ndk and alters its substrate specificity. Given the ubiquitous nature of Ndk in the living cell and its role in maintaining correct ratios of intracellular nucleoside triphosphates, the implications of the occurrence of these complexes have been discussed in relation to the precursor pool for cell wall biosynthesis as well as RNA/DNA synthesis.