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Journal of Bacteriology, July 2006, p. 5220-5227, Vol. 188, No. 14
0021-9193/06/$08.00+0     doi:10.1128/JB.00286-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

CYP153A6, a Soluble P450 Oxygenase Catalyzing Terminal-Alkane Hydroxylation

Enrico G. Funhoff,1 Ulrich Bauer,1 Inés García-Rubio,2 Bernard Witholt,1 and Jan B. van Beilen1*

Institute of Biotechnology,1 Laboratory of Physical Chemistry, Swiss Federal Institute of Technology Zürich, CH-8093 Zürich, Switzerland2

Received 24 February 2006/ Accepted 8 May 2006

The first and key step in alkane metabolism is the terminal hydroxylation of alkanes to 1-alkanols, a reaction catalyzed by a family of integral-membrane diiron enzymes related to Pseudomonas putida GPo1 AlkB, by a diverse group of methane, propane, and butane monooxygenases and by some membrane-bound cytochrome P450s. Recently, a family of cytoplasmic P450 enzymes was identified in prokaryotes that allow their host to grow on aliphatic alkanes. One member of this family, CYP153A6 from Mycobacterium sp. HXN-1500, hydroxylates medium-chain-length alkanes (C6 to C11) to 1-alkanols with a maximal turnover number of 70 min–1 and has a regiospecificity of ≥95% for the terminal carbon atom position. Spectroscopic binding studies showed that C6-to-C11 aliphatic alkanes bind in the active site with Kd values varying from ~20 nM to 3.7 µM. Longer alkanes bind more strongly than shorter alkanes, while the introduction of sterically hindering groups reduces the affinity. This suggests that the substrate-binding pocket is shaped such that linear alkanes are preferred. Electron paramagnetic resonance spectroscopy in the presence of the substrate showed the formation of an enzyme-substrate complex, which confirmed the binding of substrates observed in optical titrations. To rationalize the experimental observations on a molecular scale, homology modeling of CYP153A6 and docking of substrates were used to provide the first insight into structural features required for terminal alkane hydroxylation.

* Corresponding author. Mailing address: Institute of Biotechnology, Swiss Federal Institute of Technology Zürich, Wolfgang-Pauli-Strasse 16, CH-8093 Zürich, Switzerland. Phone: 41-1-6333444. Fax: 41-1-6331051. E-mail: vanbeilen{at}biotech.biol.ethz.ch.

Journal of Bacteriology, July 2006, p. 5220-5227, Vol. 188, No. 14
0021-9193/06/$08.00+0     doi:10.1128/JB.00286-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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