CYP153A6, a Soluble P450 Oxygenase Catalyzing Terminal-Alkane Hydroxylation

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 ϳ20 nM to 3.7 M. 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.

Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’

“…The soluble haem-containing CYP153A6 alkane hydroxylase from Mycobacterium sp. HXN-1500 has been reported to have low K d values (20 nM) for C 9 -C 11 growth substrates; however, K d values for cyclic hydrocarbons are nearly 200-fold higher (Funhoff et al, 2006). Despite the large Kinetic characterization of butane monooxygenase substrate ranges for these alkane monooxygenases, these data, together with our kinetic analyses of sBMO, emphasize the importance for these alkane monooxygenases to maintain high specificities for physiological substrates in order to out-compete oxidation of molecules that would not provide the carbon and energy needs to sustain cell growth.…”

Endophytic root bacteria associated with the natural vegetation growing at the hydrocarbon-contaminated Bitumount Provincial Historic site

“…Many alkane degraders are known to possess more than 1 alkane hydroxylase with overlapping substrate ranges (van Beilen and Funhoff 2007). Pseudomonas putida and Pseudomonas fluorescens have been demonstrated to functionally express 8 CYP153 genes along with a CYP153 family ferredoxin and ferredoxin reductase protein, allowing the host to use a wide range of substrates as carbon and energy sources (Funhoff et al 2006). Sphingomonas sp.…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…Among eukaryotes, yeasts are well-known for their ability to grow on alkanes by virtue of their membrane-bound microsomal P450s belonging CYP52 family [200][201][202][203] and they may have an important role in the biodegradation of alkanes in some oil-contaminated sites [204]. By contrast, only limited evidence showed the presence of P450 cytochromes involved in n-alkane metabolism in bacteria [30].…”

“…HXN-1500 was purified and showed the ability to hydroxylate C 6 -C 11 alkanes to 1-alkanols with high affinity and regio-selectivity [200]. Furthermore, the same primers were used to carry out a search for CYP153 genes in alkane-degrading strains that possessed AlkB-like hydroxylases only acting on long-chain length alkanes, even though these strains were also able to grow well on the medium-chain hydrocarbons [208].…”

“…These primers were used to clone the Mycobacterium sp. strain HXN-1500CYP153A6 gene and flanking regions [200]. The cytochrome P450 from Mycobacterium sp.…”