Structural basis for catalytic activity and enzyme polymerization of phospholipid hydroperoxide glutathione peroxidase-4 (GPx4).
Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a moonlighting selenoprotein, which has been implicated in anti-oxidative defense, sperm development, and cerebral embryogenesis. Among GPx-isoforms, GPx4 is unique because of its capability to reduce complex lipid hydroperoxides and its tendency toward polymerization, but the structural basis for these properties remained unclear. To address this, we solved the crystal structure of the catalytically active U46C mutant of human GPx4 to 1.55 A resolution. X-ray data indicated a monomeric protein consisting of four alpha-helices and seven beta-strands. GPx4 lacks a surface exposed loop domain, which appears to limit the accessibility of the active site of other GPx-isoforms, and these data may explain the broad substrate specificity of GPx4. The catalytic triad (C46, Q81, and W136) is localized at a flat impression of the protein surface extending into a surface exposed patch of basic amino acids (K48, K135, and R152) that also contains polar T139. Multiple mutations of the catalytic triad indicated its functional importance. Like the wild-type enzyme, the U46C mutant exhibits a strong tendency toward protein polymerization, which was prevented by reductants. Site-directed mutagenesis suggested involvement of the catalytic C46 and surface exposed C10 and C66 in polymer formation. In GPx4 crystals, these residues contact adjacent protein monomers.
Scheerer
Patrick
P
Institute of Biochemistry, University Medicine Berlin-Charité, Monbijoustr. 2, D-10117 Berlin, Germany.
Borchert
Astrid
A
Krauss
Norbert
N
Wessner
Helga
H
Gerth
Christa
C
Höhne
Wolfgang
W
Kuhn
Hartmut
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001704:Biopolymers / Q000096:biosynthesis* / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013329:Structure-Activity Relationship; D000094765:Glutathione Peroxidase GPX1
D001704:Biopolymers; D007527:Isoenzymes; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase; D003545:Cysteine; D000094765:Glutathione Peroxidase GPX1; C000722067:GPX1 protein, human