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

Abstract

Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase and phospholipase A 2 (PLA 2 ) 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 a-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 3 10 6 M 21 s 21 and a dissociation constant of 0.36 mM. Both binding and PLA 2 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 PLA 2 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.-Manevich, Y., K. S. Reddy, T. Shuvaeva, S. I. Feinstein, and A. B. Fisher. Structure and phospholipase function of peroxiredoxin 6: identification of the catalytic triad and its role in phospholipid substrate binding. J. Lipid Res.

Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase

“…We observed that phospholipase activity-dead S32A Prx6 mutant considerably lost its antiapoptotic activity. As this mutant shows a marked change of secondary structure and protein folding, 30 the role of phospholipase activity of Prx6 in TRAIL-induced cell death is not clear yet.…”

Peroxiredoxin 6 Phosphorylation and Subsequent Phospholipase A2 Activity Are Required for Agonist-mediated Activation of NADPH Oxidase in Mouse Pulmonary Microvascular Endothelium and Alveolar Macrophages

“…Expression of the C47S Prdx6 mutant partially rescued (ϳ50%) both the agonist-induced PLA 2 activity and ROS generation (Table 4). On the other hand, there was no significant rescue of either PLA 2 activity or ROS generation in endothelial cells transfected with constructs that express S32A, H26A, or D140A mutant Prdx6; these 3 amino acids constitute the Prdx6 PLA 2 catalytic triad, and each is necessary for PLA 2 activity but not for H 2 O 2 peroxidase activity (21,22). These results for "rescue" show that the PLA 2 activity of Prdx6 is required for Ang II-mediated activation of NOX2 in mouse PMVEC.…”

The serpentine path to a novel mechanism-based inhibitor of acute inflammatory lung injury

“…An Inhibitor of Acute Lung Injury • Fisher AB acid mutagenesis based on the crystal structure of the protein (17) revealed that surface-located amino acids located around the S32 are important for binding of the protein to the phospholipid substrate (58). We have proposed that the insertion of the sn-2 fatty acid of phospholipids into a narrow crypt positions it for either reduction (peroxidase activity) or hydrolysis (PLA 2 activity) (Fig.…”

“…[Reproduced with permission from Manevich et al (58).] An Inhibitor of Acute Lung Injury • Fisher AB acid mutagenesis based on the crystal structure of the protein (17) revealed that surface-located amino acids located around the S32 are important for binding of the protein to the phospholipid substrate (58).…”

“…We have carried out extensive studies of Prdx6 binding to phospholipids and determined that this feature can account for the unique Prdx6 activities (26,58,59). Site-directed amino (59).…”