Submitted on June 29, 2007
Accepted on July 23, 2007
Structure and phospholipase function of peroxiredoxin 6: Identification of the catalytic triad and its role in phospholipid substrate binding
Yefim Manevich, Konda S. Reddy, Tea Shuvaeva, Sheldon Feinstein, and Aron Fisher
Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA 19104
Corresponding Author: abf{at}mail.med.upenn.edu
Peroxiredoxin 6 (Prdx6), a bifunctional protein with glutathione peroxidase (GPx) and phospholipase A2 (PLA2) activities, is the only member of the peroxiredoxin family with the ability to hydrolyze phospholipids. Based on the identification of a potential catalytic triad from the crystal structure, site-specific mutations (S32A, H26A, D140A) of Prdx6 were used to evaluate the role of these residues in protein structure and function. Mutation of S32 converted Prdx6 to a completely -helical protein, while mutation of H26 and D140 had essentially no effect on secondary structure as detected by cellular dichroism (CD) analysis. Analysis of lipid binding by wild type Prdx6 using N-DNS-PE- or bisPyrPC-labeled small unilamellar liposomes containing a negatively charged lipid showed apparent k1=11.2x106M-1s-1 and apparent Kd=0.36 M. Both binding and PLA2 activity were abolished by S32 and H26 mutations while D140 mutation abolished activity but binding was unaffected. Overoxidation of the peroxidatic C47 to a sulfinic acid had no effect on protein binding or PLA2 activity. Using fluorescence resonance energy transfer (FRET) from protein tryptophanyls to N-DNS-PE and bisPyr(2-Pyr)-PC probes, the phospholipid polar head was shown to bind in close proximity to S32. The present results indicate that H26 is a site for interfacial binding to the liposomal surface, that S32 has a key role in both maintaining Prdx6 structure and for phospholipid substrate binding, and that D140 is involved in catalysis. Thus, this putative PLA2 catalytic triad plays an essential role for interactions of Prdx6 with phospholipid substrate which appears to optimize the protein-substrate complex for hydrolysis.
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