title: Role of the general base Glu-268 in nitroglycerin bioactivation and superoxide formation by aldehyde dehydrogenase-2.
abstract: Mitochondrial aldehyde dehydrogenase-2 (ALDH2) plays an essential role in nitroglycerin (GTN) bioactivation, resulting in formation of NO or a related activator of soluble guanylate cyclase. ALDH2 denitrates GTN to 1,2-glyceryl dinitrate and nitrite but also catalyzes reduction of GTN to NO. To elucidate the relationship between ALDH2-catalyzed GTN bioconversion and established ALDH2 activities (dehydrogenase, esterase), we compared the function of the wild type (WT) enzyme with mutants lacking either the reactive Cys-302 (C302S) or the general base Glu-268 (E268Q). Although the C302S mutation led to >90% loss of all enzyme activities, the E268Q mutant exhibited virtually unaffected rates of GTN denitration despite low dehydrogenase and esterase activities. The nucleotide co-factor NAD caused a pronounced increase in the rates of 1,2-glyceryl dinitrate formation by WT-ALDH2 but inhibited the reaction catalyzed by the E268Q mutant. GTN bioactivation measured as activation of purified soluble guanylate cyclase or release of NO in the presence of WT- or E268Q-ALDH2 was markedly potentiated by superoxide dismutase, suggesting that bioavailability of GTN-derived NO is limited by co-generation of superoxide. Formation of superoxide was confirmed by determination of hydroethidine oxidation that was inhibited by superoxide dismutase and the ALDH2 inhibitor chloral hydrate. E268Q-ALDH2 exhibited approximately 50% lower rates of superoxide formation than the WT enzyme. Our results suggest that Glu-268 is involved in the structural organization of the NAD-binding pocket but is not required for GTN denitration. ALDH2-catalyzed superoxide formation may essentially contribute to oxidative stress in GTN-exposed blood vessels.
authors.affiliation: Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, A-8010 Graz, Austria.
authors.forename: M Verena
authors.initials: MV
authors.lastname: Wenzl
authors.forename: Matteo
authors.initials: M
authors.lastname: Beretta
authors.forename: Antonius C F
authors.initials: AC
authors.lastname: Gorren
authors.forename: Andreas
authors.initials: A
authors.lastname: Zeller
authors.forename: Pravas K
authors.initials: PK
authors.lastname: Baral
authors.forename: Karl
authors.initials: K
authors.lastname: Gruber
authors.forename: Michael
authors.initials: M
authors.lastname: Russwurm
authors.forename: Doris
authors.initials: D
authors.lastname: Koesling
authors.forename: Kurt
authors.initials: K
authors.lastname: Schmidt
authors.forename: Bernd
authors.initials: B
authors.lastname: Mayer
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D000818:Animals; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D006162:Guanylate Cyclase / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000737:chemistry; D009243:NAD / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D005996:Nitroglycerin / Q000031:analogs & derivatives / Q000378:metabolism*; D017354:Point Mutation; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification / Q000378:metabolism; D000071756:Soluble Guanylyl Cyclase; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
chemical_list: D018160:Receptors, Cytoplasmic and Nuclear; D009243:NAD; D013481:Superoxides; C014509:dinitroglycerol; D009569:Nitric Oxide; D013482:Superoxide Dismutase; C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D005996:Nitroglycerin