Bacterial expression, folding, purification and characterization of soluble NTPDase5 (CD39L4) ecto-nucleotidase.

The ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) are a family of enzymes that control the levels of extracellular nucleotides, thereby modulating purinergically controlled physiological processes. Six of the eight known NTPDases are membrane-bound enzymes; only NTPDase 5 and 6 can be released as soluble enzymes. Here we report the first bacterial expression and refolding of soluble human NTPDase5 from inclusion bodies. The results show that NTPDase5 requires the presence of divalent cations (Mg2+ or Ca2+) for activity. Positive cooperativity with respect to hydrolysis of its preferred substrates (GDP, IDP and UDP) is observed, and this positive cooperativity is attenuated in the presence of nucleoside monophosphate products (e.g., GMP and AMP). In addition, comparing the biochemical properties of wild-type NTPDase5 and those of a mutant NTPDase5 (C15S, which lacks the single, non-conserved cysteine residue), also expressed in bacteria, suggests that Cys15 is not essential for either proper refolding or enzymatic activity (indicating this residue is not involved in a disulfide bond). Moreover, the substrate profile of bacterially expressed NTPDase5, as well as the C15S mutant, was determined to be similar to that of full-length, membrane-bound and soluble NTPDase5 expressed in mammalian COS cells.

Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3).

Cell membrane-bound ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are homooligomeric, with native quaternary structure required for maximal enzyme activity. In this study, we mutated lysine 79 in human ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3). The residue corresponding to lysine 79 in NTPDase3 is conserved in all known cell surface membrane NTPDases (NTPDase1, 2, 3, and 8), but not in the soluble, monomeric NTPDases (NTPDase5 and 6), or in the intracellular, two transmembrane NTPDases (NTPDase4 and 7). This conserved lysine is located between apyrase conserved region 1 (ACR1) and an invariant glycosylation site (N81), in a region previously hypothesized to be important for NTPDase3 oligomeric structure. This lysine residue was mutated to several different amino acids, and all mutants displayed substantially decreased nucleotidase activities. A basic amino acid at this position was found to be important for the increase of nucleotidase activity observed after treatment with the lectin, concanavalin A. After solubilization with Triton X-100, mutants showed little or no decrease in activity, unlike the wild-type enzyme, suggesting that the lysine at this position may be important for maintaining proper folding and for stabilizing the quaternary structure. However, mutation at this site did not result in global changes in tertiary or quaternary structure as measured by Cibacron blue binding, chemical cross linking, and native gel electrophoretic analysis, leaving open the possibility of other mechanisms by which mutation of this conserved lysine residue might decrease enzyme activity.

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

The ectonucleoside triphosphate diphosphohydrolases (NTPDases) control extracellular nucleotide concentrations, thereby modulating many important biological responses, including blood clotting and pain perception. 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. Therefore, we report here the bacterial expression, refolding, purification, and biochemical characterization of the soluble portion of human NTPDase6. 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. The K(m) for GDP (130 +/- 30 microM) is similar to that determined for the soluble rat NTPDase6 expressed in mammalian cells. 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. Four of the five cysteine residues in the soluble NTPDase6 are highly conserved among all the NTPDases, while the fifth residue is not. 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. 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. Due to conservation of these cysteine residues, these two disulfide bonds are likely to exist in all NTPDases. A structural model for NTPDase6, incorporating these and other findings obtained with other NTPDases, is proposed.