Abstract
Studies with the fruit-fly Drosophila melanogaster demonstrated that the enzyme sniffer prevented oxidative stress-induced neurodegeneration. Mutant flies overexpressing sniffer had significantly extended life spans in a 99.5% oxygen atmosphere compared to wild-type flies. However, the molecular mechanism of this protection remained unclear. Sequence analysis and database searches identified sniffer as a member of the short-chain dehydrogenase/reductase superfamily with a 27.4% identity to the human enzyme carbonyl reductase type I (CBR1). As CBR1 catalyzes the reduction of the lipid peroxidation products 4HNE and 4ONE, we tested whether sniffer is able to metabolize these lipid derived aldehydes by carbonyl reduction. To produce recombinant enzyme, the coding sequence of sniffer was amplified from a cDNA-library, cloned into a bacterial expression vector and the His-tagged protein was purified by Ni-chelate chromatography. We found that sniffer catalyzed the NADPH-dependent carbonyl reduction of 4ONE (K(m)=24±2 μM, k(cat)=500±10 min(-1), k(cat)/K(m)=350 s(-1) mM(-1)) but not that of 4HNE. The reaction product of 4ONE reduction by sniffer was mainly 4HNE as shown by HPLC- and GC/MS analysis. Since 4HNE, though still a potent electrophile, is less neurotoxic and protein reactive than 4ONE, one mechanism by which sniffer exerts its neuroprotective effects in Drosophila after oxidative stress may be enzymatic reduction of 4ONE.
