title: Amino acids involved in polyphosphate synthesis and its mobilization are distinct in polyphosphate kinase-1 from Mycobacterium tuberculosis.
abstract: BACKGROUND
  In bacteria polyphosphates (poly-P) are involved in cellular metabolism and development especially during stress. The enzyme, principally involved in polyphosphate biosynthesis and its mobilization leading to generation of NTPs, is known as polyphosphate kinase (PPK).
  
  
  RESULTS
  Among two genes of polyphosphate kinases present in Mycobacterium tuberculosis, we cloned and expressed PPK1 in Escherichia coli as histidine-tagged protein. This ∼86 kDa protein is capable of autophosphorylation and synthesis of poly-P as well as NTP. Among 22 conserved histidine residues, we found only His-491 is autophosphorylated and crucial for any enzymatic activity. Substitution of His-510 caused mPPK1 protein deficient but not defective in autophosphorylation, thereby contrary to earlier reports negating any role of this residue in the process. However, mutation of His-510 with either Ala or Gln affected ATP or poly-P synthesis depending on the substitution; while such effects were severe with H510A but mild with H510Q. Furthermore, mPPK1 also renders auxiliary nucleotide diphosphate kinase function by synthesizing virtually all NTPs/dNTPs from their cognate NDPs/dNDPs by utilizing poly-P as the phosphate donor albeit with varied efficiency. To assess the influence of other catalytic domain residues of mPPK1 towards its functionality, we designed mutations based on E. coli PPK1 crystal structure since it owes 68% amino acid sequence similarity with mPPK1. Interestingly, our results revealed that mutations in mPPK1 affecting poly-P synthesis always affected its ATP synthesizing ability; however, the reverse may not be true.
  
  
  CONCLUSIONS
  We conclude that amino acid residues involved in poly-P and ATP synthesizing activities of mPPK1 are distinct. Considering conserved nature of PPK1, it seems our observations have broader implications and not solely restricted to M. tuberculosis only.
authors: Council of Scientific and Industrial Research-Institute of Microbial Technology, Sector 39A, Chandigarh, India.
authors: Payal
authors: P
authors: Mittal
authors: Subramanian
authors: S
authors: Karthikeyan
authors: Pradip K
authors: PK
authors: Chakraborti
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000596:Amino Acids; D001692:Biological Transport; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000378:metabolism; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D010766:Phosphorylation; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry* / Q000378:metabolism*; D011122:Polyphosphates / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D011122:Polyphosphates; D000255:Adenosine Triphosphate; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase; D009701:Nucleoside-Diphosphate Kinase