PMID: 14529283

 

    Legend: Gene, Sites

Title : Bacterial expression, characterization, and disulfide bond determination of soluble human NTPDase6 ( CD39L2 ) nucleotidase : implications for structure and function

Abstract :
  1. The ectonucleoside triphosphate diphosphohydrolases (NTPDases) control extracellular nucleotide concentrations, thereby modulating many important biological responses, including blood clotting and pain perception
  2. NTPDases1-4 are oligomeric integral membrane proteins , whereas NTPDase5 ( CD39L4 ) and NTPDase6 ( CD39L2 ) are soluble monomeric enzymes , making them more amenable to thorough structural and functional analyses than the membrane-bound forms
  3. Therefore, we report here the bacterial expression, refolding, purification, and biochemical characterization of the soluble portion of human NTPDase6
  4. Consistent with the enzyme expressed in mammalian cells, this recombinant NTPDase6 efficiently hydrolyzes GDP, IDP , and UDP (specific activity of approximately 50000 micromol mg(-1) h(-1)), with slower hydrolysis of CDP , ITP, GTP, CTP , ADP, and UTP and virtually no hydrolysis of ATP
  5. The K(m) for GDP (130 +/- 30 microM) is similar to that determined for the soluble rat NTPDase6 expressed in mammalian cells
  6. The secondary structure of the refolded enzyme was determined by circular dichroism to be 33% alpha-helix, 18% beta-sheet, and 49% random coil, consistent with the secondary structure predicted from the amino acid sequence of soluble NTPDase6
  7. Four of the five cysteine residues in the soluble NTPDase6 are highly conserved among all the NTPDases, while the fifth residue is not
  8. Mutation of this nonconserved cysteine resulted in an enzyme very similar to wild type in its enzymology and secondary structure, indicating that this cysteine exists as a free sulfhydryl and is not essential for structure or function
  9. The disulfide pairing of the other four cysteine residues was determined as Cys(249)-Cys(280) and Cys(340)-Cys(354) by HPLC and mass spectral analysis of tryptic peptides
  10. Due to conservation of these cysteine residues , these two disulfide bonds are likely to exist in all NTPDases
  11. A structural model for NTPDase6 , incorporating these and other findings obtained with other NTPDases, is proposed
Output (sent_index, trigger, protein, sugar, site):
Output(Part-Of) (sent_index, protein, site):
  • 6. NTPDase6, sequence
  • 7. NTPDase6, cysteine residues
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX
NTPDase5 957 2
NTPDase6 85260 0,2,3,4,5,6,7,11
CDP 1523 4
CD39L2 955 0,2
CD39L4 957 2