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Elife July 6, 2016; 5
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The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity.

Iordanov I , Mihályi C , Tóth B , Csanády L .

Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9. When expressed in E.coli, chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified. In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity (kcat~4-9s(-1)), that of NUDT9H did not support catalysis. Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating (K1/2~1-5 μM), confirming functionality of chimeric domains. Thus, TRPM2 is not a ''chanzyme''. Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies.

PubMed ID: 27383051
PMC ID: PMC4974056
Article link: Elife
Grant support: [+]

Species referenced: Xenopus
Genes referenced: ins nudt9 rb1 rbl2 sia2 trpm2

Article Images: [+] show captions
References [+] :
Bessman, The MutT proteins or "Nudix" hydrolases, a family of versatile, widely distributed, "housecleaning" enzymes. 1996, Pubmed