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Journal of Lipid Research, Vol. 48, 2306-2318, October 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Structure and phospholipase function of peroxiredoxin 6: identification of the catalytic triad and its role in phospholipid substrate binding¹

Yefim Manevich^*, Konda S. Reddy, Tea Shuvaeva^*, Sheldon I. Feinstein^* and Aron B. Fisher^2,*

^* Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104
Department of Biochemistry and Biophysics, University of Pennsylvania Medical Center, Philadelphia, PA 19104

¹ This work was presented in part at the Experimental Biology meetings in April 2004 (Washington, DC) and April 2005 (San Diego, CA).

Published, JLR Papers in Press, July 24, 2007.

² To whom correspondence should be addressed. e-mail: abf{at}mail.med.upenn.edu

Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase and phospholipase A₂ (PLA₂) activities, and it alone among mammalian peroxiredoxins can hydrolyze phospholipids. After identifying a potential catalytic triad (S32, H26, D140) from the crystal structure, site-specific mutations were used to evaluate the role of these residues in protein structure and function. The S32A mutation increased Prdx6 -helical content, whereas secondary structure was unchanged by mutation to H26A and D140A. Lipid binding by wild-type Prdx6 to negatively charged unilamellar liposomes showed an apparent rate constant of 11.2 x 10⁶ M^–1 s^–1 and a dissociation constant of 0.36 µM. Both binding and PLA₂ activity were abolished in S32A and H26A; in D140A, activity was abolished but binding was unaffected. Overoxidation of the peroxidatic C47 had no effect on lipid binding or PLA₂ activity. Fluorescence resonance energy transfer from endogenous tryptophanyls to lipid probes showed binding of the phospholipid polar head in close proximity to S32. Thus, H26 is a site for interfacial binding to the liposomal surface, S32 has a key role in maintaining Prdx6 structure and for phospholipid substrate binding, and D140 is involved in catalysis. This putative catalytic triad plays an essential role for interactions of Prdx6 with phospholipid substrate to optimize the protein-substrate complex for hydrolysis.

Supplementary key words liposomes • circular dichroism • fluorescence resonance energy transfer • site-directed mutagenesis • lipid binding • peroxidase

Abbreviations: bisPyr-PC, 1,2-bis(1-pyrenedecanoyl)-sn-glycero-phosphocholine; CD, circular dichroism; DPPC, dipalmitoyl phosphatidylcholine; FRET, fluorescence resonance energy transfer; [³H]DPPC, 1-palmitoyl,[9,10-³H]2-palmitoyl sn-glycero-3-phosphocholine; MJ33, 1-hexadecyl-3-trifluoroethyl-sn-glycero-2-phosphomethanol; N-DNS-PE, N-(5-dimethylaminonaphthalene-1-sulfonyl)-sn-glycero-3-phosphoethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PLA₂, phospholipase A₂; PLPC, 1-palmitoyl,2-linolenoyl-sn-glycero-3-phosphatidylcholine; Prdx6, peroxiredoxin 6; PS, phosphatidylserine; 2Pyr-PC, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine

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