1-Cys peroxiredoxin, a bifunctional enzyme with glutathione peroxidase andphospholipase A2 activities

This report provides definitive evidence that the protein 1-Cys peroxiredox in is a bifunctional ("moonlighting") enzyme with two distinct active sites . We have previously shown that human, rat, and bovine lungs contain an aci dic Ca2+-independent phospholipase A(2) (aiPLA(2)). The cDNA encoding aiPLA (2) was found to be identical to that of a non-selenium glutathione peroxid ase (NSGPx). Protein expressed using a previously reported E, coli construc t which has a His-tag and 50 additional amino acids at the NH2 terminus, di d not exhibit aiPLA(2) activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escheric hia coli generated a protein that hydrolyzed the sn-2 acyl chain of phospho lipids at pH 4, and exhibited NSGPx activity with H2O2 at pH 8. The express ed 1-Cys peroxiredoxin has identical functional properties to the native lu ng enzyme: aiPLA(2) activity is inhibited by the serine protease inhibitor, diethyl p-nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-tr ifluoroethylglycero-sn-2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx ac tivity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys perox iredoxin mAb 8B3 antibody which have no effect on aiPLA(2) activity. Mutati on of Ser(32) to Ala abolishes aiPLA(2) activity, yet the NSGPx activity re mains unaffected; a Cys(47) to Ser mutant is devoid of peroxidase activity but aiPLA(2) activity remains intact. These results suggest that Ser32 in t he GDSWG consensus sequence provides the catalytic nucleophile for the hydr olase activity of aiPLA(2) while Cys47 in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic prop erties of 1-Cys peroxiredoxin are compatible with a simultaneous role for t he protein in the regulation of phospholipid turnover as well as in protect ion against oxidative injury.