CYP153A6, a Soluble P450 Oxygenase Catalyzing Terminal-Alkane Hydroxylation

Author:

Funhoff Enrico G.1,Bauer Ulrich1,García-Rubio Inés2,Witholt Bernard1,van Beilen Jan B.1

Affiliation:

1. Institute of Biotechnology

2. Laboratory of Physical Chemistry, Swiss Federal Institute of Technology Zürich, CH-8093 Zürich, Switzerland

Abstract

ABSTRACT 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 (C 6 to C 11 ) 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 C 6 -to-C 11 aliphatic alkanes bind in the active site with K d 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.

Publisher

American Society for Microbiology

Subject

Molecular Biology,Microbiology

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