Abstract 231: Cloning, Expression, Charecterization and Redox Regulation of Human DDAH-1: Implications in NO Regulation
Altered NO biosynthesis is known to play a central role in the pathogenesis of endothelial dysfunction. It is hypothesized that reduced NO bioavailability may result from an increase in endogenous NOS inhibitors, ADMA and NMMA, which are normally metabolized by dimethyarginine dimethylamine hydrolase (DDAH). DDAH hydrolyzes side-chain methylated L-arg and thus regulates the activity of NOS. To date, the few studies published on DDAH have been carried out using enzyme purified from pseudomonas or rat kidney homogenates. Herein we report the cloning, expression and kinetic properties of hDDAH-1 and its regulation by redox environment. Human DDAH-1 was cloned into an e. coli expression vector and recombinant hDDAH-1 purifed to > 95% purity. Kinetic studies from the enzyme demonstrated Km values of 18.2 and 27.1 μM and Vmax values of 303 and 182 nmols/mg/min for ADMA and L-NMMA, respectively. Oxidant exposure studies were performed to determine the effects of reactive oxygen and reactive nitrogen species on DDAH activity. Results demonstrated that low level oxidant exposure had little effect on enzyme activity and that concentrations approaching 1 mM were needed to confer significant enzyme inhibition. However, exposure of hDDAH-1 to lipid oxidation products, such as 4-HNE, dose dependently inhibited DDAH activity with 15% inhibition observed at 10 μM, 50% inhibition at 50 μM and near complete inhibition at 100 μM. These levels represent pathophysiological concentrations of this lipid peroxidation product and suggest that DDAH activity can be impaired under conditions of increased oxidative stress. Proteomic analysis revealed that exposure of hDDAH-1 to 4-HNE results in Schiff base adduct formation of critical amino acid residues located within the enzyme active site. Thus, 4-HNE directly modifies hDDAH-1 with subsequent loss of enzyme activity. To determine whether this loss of DDAH activity results in NOS impairment, studies were performed using cultured endothelial cells. Results demonstrated that exposure of cells to 4-HNE (50 μM) inhibited eNOS derived NO production by 45% and reduced cellular DDAH activity by 41%. These results demonstrate a critical role for DDAH in the pathogenesis of endothelial dysfunction under conditions of oxidative stress.