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PEDS Advance Access published online on October 24, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm028
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Structure-activity correlations in pentachlorobenzene oxidation by engineered cytochrome P450cam1

Feng Xu2, Stephen G. Bell3, Zihe Rao2,4,5 and Luet-Lok Wong3,5

2Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Tsinghua University, Beijing 100084, China 3Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, UK 4National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

5 To whom correspondence should be addressed. E-mail: raozh{at}xtal.tsinghua.edu.cn (Z.R.); E-mail: luet.wong{at}chem.ox.ac.uk (L.-L.W.)

We had reported engineering of the heme monooxygenase cytochrome P450cam from Pseudomonas putida with the F87W/Y96F/L244A/V247L mutations for the oxidation of pentachlorobenzene (PeCB), a recalcitrant environmental contaminant, to pentachlorophenol. In order to provide further insights into P450 structure, function and substrate recognition, we have determined the crystal structure of this 4-mutant without a substrate and its complex with PeCB. PeCB is bound face-on to the heme, with a weak Fe—Cl interaction. One PeCB chlorine is located in the cavity generated by the L244A mutation, in striking illustration of the role of this mutation in promoting PeCB binding. The structures also show that the P450cam oxygen-binding groove between G248 and T252 is flexible and can tolerate significant deviations from their conformations in the wild type without loss of enzyme activity. Analysis of the PeCB binding interactions led to introduction of the T101A mutation to enable the substrate to reorient during the catalytic cycle for more efficient oxidation. The resultant 5-mutant F87W/Y96F/T101A/L244A/V247L is 3-fold more active for PeCB oxidation than the 4-mutant. Polychlorinated benzene binding by the mutants and the partitioning between substrate oxidation and non-productive (uncoupling) side reactions are correlated with the structural data.

Keywords: biodegradation/cytochrome P450/mutagenesis/polychlorinated aromatics/protein engineering

Received March 22, 2007; revised May 12, 2007; accepted May 15, 2007.

1 The coordinates for the structure of the F87W/Y96F/L244A/V247L mutant complexed with pentachlorobenzene have been deposited in the Protein Data Bank (access code 2GQX).


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