Crystal structure of a ferredoxin reductase for the CYP199A2 system from Rhodopseudomonas palustris
Correction(s) for this article
-
Correction to the article “Crystal Structure of a Ferredoxin Reductase for the CYP199A2 System from Rhodopseudomonas palustris”
- Volume 78Issue 2Proteins: Structure, Function, and Bioinformatics
- pages: 501-501
- First Published online: October 2, 2009
Feng Xu
Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China
F. Xu and S.G. Bell contributed equally to this work.
Search for more papers by this authorCorresponding Author
Stephen G. Bell
Department of Chemistry, University of Oxford, Oxford OX1 3QR, United Kingdom
F. Xu and S.G. Bell contributed equally to this work.
Stephen G. Bell, Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, United Kingdom===
Mark Bartlam, College of Life Sciences and Tianjin Key Laboratory of Protein Science, Nankai University, Tianjin 300071, People's Republic of China===
Search for more papers by this authorYing Peng
Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China
Search for more papers by this authorEachan O.D. Johnson
Department of Chemistry, University of Oxford, Oxford OX1 3QR, United Kingdom
Search for more papers by this authorCorresponding Author
Mark Bartlam
Center for Structural Biology, College of Life Sciences and Tianjin Key Laboratory of Protein Science, Nankai University, Tianjin 300071, People's Republic of China
Stephen G. Bell, Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, United Kingdom===
Mark Bartlam, College of Life Sciences and Tianjin Key Laboratory of Protein Science, Nankai University, Tianjin 300071, People's Republic of China===
Search for more papers by this authorZihe Rao
Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China
Center for Structural Biology, College of Life Sciences and Tianjin Key Laboratory of Protein Science, Nankai University, Tianjin 300071, People's Republic of China
Search for more papers by this authorLuet-Lok Wong
Department of Chemistry, University of Oxford, Oxford OX1 3QR, United Kingdom
Search for more papers by this authorFeng Xu
Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China
F. Xu and S.G. Bell contributed equally to this work.
Search for more papers by this authorCorresponding Author
Stephen G. Bell
Department of Chemistry, University of Oxford, Oxford OX1 3QR, United Kingdom
F. Xu and S.G. Bell contributed equally to this work.
Stephen G. Bell, Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, United Kingdom===
Mark Bartlam, College of Life Sciences and Tianjin Key Laboratory of Protein Science, Nankai University, Tianjin 300071, People's Republic of China===
Search for more papers by this authorYing Peng
Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China
Search for more papers by this authorEachan O.D. Johnson
Department of Chemistry, University of Oxford, Oxford OX1 3QR, United Kingdom
Search for more papers by this authorCorresponding Author
Mark Bartlam
Center for Structural Biology, College of Life Sciences and Tianjin Key Laboratory of Protein Science, Nankai University, Tianjin 300071, People's Republic of China
Stephen G. Bell, Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, United Kingdom===
Mark Bartlam, College of Life Sciences and Tianjin Key Laboratory of Protein Science, Nankai University, Tianjin 300071, People's Republic of China===
Search for more papers by this authorZihe Rao
Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China
Center for Structural Biology, College of Life Sciences and Tianjin Key Laboratory of Protein Science, Nankai University, Tianjin 300071, People's Republic of China
Search for more papers by this authorLuet-Lok Wong
Department of Chemistry, University of Oxford, Oxford OX1 3QR, United Kingdom
Search for more papers by this authorAbstract
Cytochrome P450-199A2 from Rhodopseudomonas palustris oxidizes para-substituted benzoic acids and may play a role in lignin and aromatic acid degradation pathways in the bacterium. CYP199A2 has an associated [2Fe-2S] ferredoxin, palustrisredoxin (Pux) but not a ferredoxin reductase. A genome search identified the palustrisredoxin reductase (PuR) gene. PuR was produced in Escherichia coli and shown to be a flavin-dependent protein that supports efficient electron transfer from NADH to Pux, thus reconstituting CYP199A2 monooxygenase activity (kcat = 37.9 s–1 with 4-methoxybenzoic acid). The reduction of Pux by PuR shows Km = 4.2 μM and kcat = 262 s–1 in 50 mM Tris, pH 7.4. Km is increased to 154 μM in the presence of 200 mM KCl, indicating the importance of ionic interactions in PuR/Pux binding. The crystal structure of PuR has been determined at 2.2 Å resolution and found to be closely related to that of other oxygenase-coupled NADH-dependent ferredoxin reductases. Residues on the surface that had been proposed to be involved in ferredoxin reductase-ferredoxin binding are conserved in PuR. However, Lys328 in PuR lies over the FAD isoalloxazine ring and, together with His11 and Gln41, render the electrostatic potential of the surface more positive and may account for the greater involvement of electrostatic interactions in ferredoxin binding by PuR. Consistent with these observations the K328G mutation weakened Pux binding and virtually eliminated the dependence of PuR/Pux binding on salt concentration, thus confirming that the FAD si side surface in the vicinity of Lys328 is the ferredoxin binding site. Proteins 2009. © 2009 Wiley-Liss, Inc.
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