Three-dimensional structure and enzymatic function of proapoptotic human p53-inducible quinone oxidoreductase PIG3.
Tumor suppressor p53 regulates the expression of p53-induced genes (PIG) that trigger apoptosis. PIG3 or TP53I3 is the only known member of the medium chain dehydrogenase/reductase superfamily induced by p53 and is used as a proapoptotic marker. Although the participation of PIG3 in the apoptotic pathway is proven, the protein and its mechanism of action were never characterized. We analyzed human PIG3 enzymatic function and found NADPH-dependent reductase activity with ortho-quinones, which is consistent with the classification of PIG3 in the quinone oxidoreductase family. However, the activity is much lower than that of zeta-crystallin, a better known quinone oxidoreductase. In addition, we report the crystallographic structure of PIG3, which allowed the identification of substrate- and cofactor-binding sites, with residues fully conserved from bacteria to human. Tyr-59 in zeta-crystallin (Tyr-51 in PIG3) was suggested to participate in the catalysis of quinone reduction. However, kinetics of Tyr/Phe and Tyr/Ala mutants of both enzymes demonstrated that the active site Tyr is not catalytic but may participate in substrate binding, consistent with a mechanism based on propinquity effects. It has been proposed that PIG3 contribution to apoptosis would be through oxidative stress generation. We found that in vitro activity and in vivo overexpression of PIG3 accumulate reactive oxygen species. Accordingly, an inactive PIG3 mutant (S151V) did not produce reactive oxygen species in cells, indicating that enzymatically active protein is necessary for this function. This supports that PIG3 action is through oxidative stress produced by its enzymatic activity and provides essential knowledge for eventual control of apoptosis.
From the Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Sergio
S
Porté
Institut de Biologia Molecular (IBMB-Consejo Superior de Investigaciones Científicas) and IRB Barcelona, Parc Científic de Barcelona, Josep-Samitier 1-5, 08028 Barcelona, Spain.
Eva
E
Valencia
From the Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Evgenia A
EA
Yakovtseva
From the Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Emma
E
Borràs
Structural Genomics Consortium, Old Road Research Campus, University of Oxford, Oxford OX3 7DQ, United Kingdom.
Naeem
N
Shafqat
Structural Genomics Consortium, Old Road Research Campus, University of Oxford, Oxford OX3 7DQ, United Kingdom.
Judit É
JÉ
Debreczeny
Structural Genomics Consortium, Old Road Research Campus, University of Oxford, Oxford OX3 7DQ, United Kingdom.
Ashley C W
ACW
Pike
Structural Genomics Consortium, Old Road Research Campus, University of Oxford, Oxford OX3 7DQ, United Kingdom; Botnar Research Center, Oxford Biomedical Research Unit, Oxford OX3 7LD, United Kingdom.
Udo
U
Oppermann
From the Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Jaume
J
Farrés
Institut de Biologia Molecular (IBMB-Consejo Superior de Investigaciones Científicas) and IRB Barcelona, Parc Científic de Barcelona, Josep-Samitier 1-5, 08028 Barcelona, Spain.
Ignacio
I
Fita
From the Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Electronic address: xavier.pares@uab.es.
Xavier
X
Parés
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology; D001665:Binding Sites; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010802:Phylogeny; D020836:Protein Structure, Quaternary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
D047908:Intracellular Signaling Peptides and Proteins; D011518:Proto-Oncogene Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; C495901:TP53 protein, human; C411296:TP53I3 protein, human; D016159:Tumor Suppressor Protein p53; D014443:Tyrosine; D009249:NADP