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J. Biol. Chem., Vol. 277, Issue 40, 37519-37526, October 4, 2002

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Crystal Structure of the F87W/Y96F/V247L Mutant of Cytochrome P-450cam with 1,3,5-Trichlorobenzene Bound and Further Protein Engineering for the Oxidation of Pentachlorobenzene and Hexachlorobenzene^*,

Xuehui Chen, Alexandra Christopher^§, Jonathan P. Jones^§, Stephen G. Bell^§, Qing Guo, Feng Xu, Zihe Rao^¶, and Luet-Lok Wong^§

From the  Laboratory of Structural Biology, Department of Biological Science and Technology & Ministry of Education Laboratory of Protein Science, Tsinghua University, Beijing 100084, China and the ^§ Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QR, United Kingdom

We reported previously that the F87W/Y96F/V247L mutant of cytochrome P-450cam (CYP101) from Pseudomonas putida catalyzed the rapid oxidation of lightly chlorinated benzenes, but pentachlorobenzene oxidation was slow (Jones, J. P., O'Hare, E. J., and Wong, L. L. (2001) Eur. J. Biochem. 268, 1460-1467). In the present work, we determined the crystal structure of this mutant with bound 1,3,5-trichlorobenzene. The substrate was bound to crystallographically independent CYP101 molecules in at least three different orientations, which were distinguished by the angle between the benzene ring and the porphyrin, and one orientation contained an Fe-Cl interaction. In another orientation, the substrate was almost parallel to the heme, with a C-H bond closest to the iron. The enzyme/substrate contacts suggested that the L244A mutation should promote the binding of pentachlorobenzene and hexachlorobenzene by creating space to accommodate the extra chlorines. The F87W/Y96F/L244A/V247L mutant thus designed was found to oxidize pentachlorobenzene at a rate of 82.5 nmol (nmol CYP101)¹ min¹, 45 times faster than the F87W/Y96F/V247L parent mutant. The rate of hexachlorobenzene oxidation was increased 200-fold, to 2.0 min¹. Both substrates are oxidized to pentachlorophenol, which is degraded by micro-organisms. In principle, the F87W/Y96F/L244A/V247L mutant could have applications in the bioremediation of polychlorinated benzenes.

The atomic coordinates and the structure factors (code 1J51) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

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