Cytochrome P450 systems--biological variations of electron transport chains.

Pollution of soil and water environments by crude oil has been, and is still today, an important problem. Crude oil is a complex mixture of thousands of compounds. Among them, alkanes constitute the major fraction. Alkanes are saturated hydrocarbons of different sizes and structures. Although they are chemically very inert, most of them can be efficiently degraded by several microorganisms. This review summarizes current knowledge on how microorganisms degrade alkanes, focusing on the biochemical pathways used and on how the expression of pathway genes is regulated and integrated within cell physiology.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1462-2920.2009.01948.x

Degradation of alkanes by bacteria

This review focuses on the role and distribution in the environment of alkane hydroxylases and their (potential) applications in bioremediation and biocatalysis. Alkane hydroxylases play an important role in the microbial degradation of oil, chlorinated hydrocarbons, fuel additives, and many other compounds. Environmental studies demonstrate the abundance of alkane degraders and have lead to the identification of many new species, including some that are (near)-obligate alkanotrophs. The availability of a growing collection of alkane hydroxylase gene sequences now allows estimations of the relative abundance of the different enzyme systems and the distribution of the host organisms.

/pdf/alkane-hydroxylases-involved-in-microbial-alkane-degradation-3ctil2prp4.pdf

Alkane hydroxylases involved in microbial alkane degradation.

P450BM3 (CYP102A1), a fatty acid hydroxylase from Bacillus megaterium, has been extensively studied over a period of almost forty years. The enzyme has been redesigned to catalyse the oxidation of non-natural substrates as diverse as pharmaceuticals, terpenes and gaseous alkanes using a variety of engineering strategies. Crystal structures have provided a basis for several of the catalytic effects brought about by mutagenesis, while changes to reduction potentials, inter-domain electron transfer rates and catalytic parameters have yielded functional insights. Areas of active research interest include drug metabolite production, the development of process-scale techniques, unravelling general mechanistic aspects of P450 chemistry, methane oxidation, and improving selectivity control to allow the synthesis of fine chemicals. This review draws together the disparate research themes and places them in a historical context with the aim of creating a resource that can be used as a gateway to the field.

P450BM3 (CYP102A1): connecting the dots

Enzyme-mediated oxidations for the chemist

A number of oxygenated monoterpenes present at low concentrations in plant oils have anticarcinogenic properties. One of the most promising compounds in this respect is ()-perillyl alcohol. Since this natural product is present only at low levels in a few plant oils, an alternative, synthetic source is desirable. Screening of 1,800 bacterial strains showed that many alkane degraders were able to specifically hydroxylate L-limonene in the 7 position to produce enantiopure ()-perillyl alcohol. The oxygenase responsible for this was purified from the best-performing wild-type strain, Mycobacterium sp. strain HXN-1500. By using N-terminal sequence information, a 6.2-kb ApaI fragment was cloned, which encoded a cytochrome P450, a ferredoxin, and a ferredoxin reductase. The three genes were successfully coexpressed in Pseudomonas putida by using the broad-host-range vector pCom8, and the recombinant converted limonene to perillyl alcohol with a specific activity of 3 U/g (dry weight) of cells. The construct was subsequently used in a 2-liter bioreactor to produce perillyl alcohol on a scale of several grams. The production of ()-perillyl alcohol from L-limonene is of interest because of the limited availability of ()-perillyl alcohol in nature and its proven anticarcinogenic properties; phase II trials to evaluate perillyl alcohol for the treatment of breast, pancreatic, and colorectal cancer are in progress (37). The only microbial enzyme system described thus far that transforms limonene to perillyl alcohol was found in Bacillus stearothermophilus BR388. However, this enzyme system is not sufficiently regiospecific; significant quantities of carveol, carvone, and terpineol are also produced (25). Removal of these side products is difficult as their boiling points and hydrophobicities are almost identical, and expensive purification methods (for example, chromatography) would be required to obtain sufficiently pure perillyl alcohol. Therefore, the industrial production of perillyl alcohol with this Bacillus enzyme system is not attractive. The conversion of limonene to perillic acid by a Pseudomonas putida strain expressing a cymene monooxygenase was described by Mars et al. (21) and could be interesting as perillyl alcohol is likely to be an intermediate in the production of perillic acid. Other literature concerning limonene biotransformations was reviewed recently (5). The approach that we used to find strains capable of regiospecific hydroxylation of limonene consisted of screening a collection of 1,800 bacterial strains grown on a range of relatively reduced substrates, such as toluene, naphthalene, and various alkanes. Using this approach, we anticipated that we would find oxygenases involved in catabolic pathways that would accept L-limonene as a substrate. Previous work has demonstrated that many catabolic oxygenases accept a wide range of unnatural substrates. Toluene dioxygenases, for ex

Biocatalytic Production of Perillyl Alcohol from Limonene by Using a Novel Mycobacterium sp. Cytochrome P450 Alkane Hydroxylase Expressed in Pseudomonas putida

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 > or =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 approximately 20 nM to 3.7 microM. 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.

Ulrich Bauer

Papers

Biocatalytic Production of Perillyl Alcohol from Limonene by Using a Novel Mycobacterium sp. Cytochrome P450 Alkane Hydroxylase Expressed in Pseudomonas putida

CYP153A6, a Soluble P450 Oxygenase Catalyzing Terminal-Alkane Hydroxylation

Hydroxylation and epoxidation reactions catalyzed by CYP153 enzymes