CYP108N14: A Monoterpene Monooxygenase from Rhodococcus globerulus.

Rhodococcus globerulus (R. globerulus) was isolated from the soil beneath a Eucalypt tree. Metabolic growth studies revealed that R. globerulus was capable of living on certain monoterpenes, including 1,8-cineole and p-cymene, as sole sources of carbon and energy. Multiple P450 genes were identified in the R. globerulus genome that shared homology to known bacterial, monoterpene hydroxylating P450s. To date, two of these P450s have been expressed and characterised as 1,8-cineole (CYP176A1) and p-cymene (CYP108N12) monooxygenases that are believed to initiate the biodegradation of these terpenes. In this work, another putative P450 gene (CYP108N14) was identified in R. globerulus genome. Given its amino acid sequence identity to other monoterpene hydroxylating P450s it was hypothesised to catalyse monoterpene hydroxylation. These include CYP108A1 from Pseudomonas sp. (47 % identity, 68 % similarity) which hydroxylates α-terpineol, and CYP108N12 also from R. globerulus (62 % identity, 77 % similarity). Also present in the operon containing CYP108N14 were putative ferredoxin and ferredoxin reductase genes, suggesting a typical Class I P450 system. CYP108N14 was successfully over-expressed heterologously and purified, resulting in a good yield of CYP108N14 holoprotein. However, neither the ferredoxin nor ferredoxin reductase could be produced heterologously. Binding studies with CYP108N14 revealed a preference for the monoterpenes p-cymene, (R)-limonene, (S)-limonene, (S)-α-terpineol and (S)-4-terpineol. An active catalytic system was reconstituted with the non-native redox partners cymredoxin (from the CYP108N12 system) and putidaredoxin reductase (from the CYP101A1 system). CYP108N14 when supported by these redox partners was able to catalyse the hydroxylation of the five aforementioned substrates selectively at the methyl benzylic/allylic positions.

Cymredoxin, a [2Fe-2S] ferredoxin, supports catalytic activity of the p-cymene oxidising P450 enzyme CYP108N12.

Rhodococcus globerulus is a metabolically active organism that has been shown to utilise eucalypt oil as its sole source of carbon and energy. This oil includes 1,8-cineole, p-cymene and limonene. Two identified and characterised cytochromes P450 (P450s) from this organism initiate the biodegradation of the monoterpenes 1,8-cineole (CYP176A1) and p-cymene (CYP108N12). Extensive characterisation has been completed for CYP176A1 and it has been successfully reconstituted with its immediate redox partner, cindoxin, and E. coli flavodoxin reductase. Two putative redox partner genes are encoded in the same operon as CYP108N12 and here the isolation, expression, purification, and characterisation of its specific [2Fe-2S] ferredoxin redox partner, cymredoxin is presented. Reconstitution of CYP108N12 with cymredoxin in place of putidaredoxin, a [2Fe-2S] redox partner of another P450, improves both the rate of electron transfer (from 13 ± 2 to 70 ± 1 µM NADH/min/µM CYP108N12) and the efficiency of NADH utilisation (the so-called coupling efficiency increases from 13% to 90%). Cymredoxin improves the catalytic ability of CYP108N12 in vitro. Aldehyde oxidation products of the previously identified substrates p-cymene (4-isopropylbenzaldehyde) and limonene (perillaldehyde) were observed in addition to major hydroxylation products 4-isopropylbenzyl alcohol and perillyl alcohol respectively. These further oxidation products had not previously been seen with putidaredoxin supported oxidation. Furthermore, when supported by cymredoxin CYP108N12 is able to oxidise a wider range of substrates than previously reported. These include o-xylene, α-terpineol, (-)-carveol and thymol yielding o-tolylmethanol, 7-hydroxyterpineol, (4R)-7-hydroxycarveol and 5-hydroxymethyl-2-isopropylphenol, respectively. Cymredoxin is also capable of supporting CYP108A1 (P450terp) and CYP176A1 activity, allowing them to catalyse the hydroxylation of their native substrates α-terpineol to 7-hydroxyterpineol and 1,8-cineole to 6ß-hydroxycineole respectively. These results indicate that cymredoxin not only improves the catalytic capability of CYP108N12 but can also support the activity of other P450s and prove useful for their characterisation.

Synthesis of substituted norcaranes for use as probes of enzyme mechanisms.

Norcarane is a mechanistic probe of monooxygenase enzymes that is able to detect the presence of cationic or radical intermediates. The addition of substituents around the bicycloheptane ring of the norcarane scaffold can assist in improving enzyme binding affinity and thus improve the regioselectivity of oxidation. Here we prepare in three-step, diastereoselective syntheses, ten norcaranes monosubstituted α to the cyclopropane as advanced probes. Four of these compounds were examined in enzyme binding experiments to evaluate their potential as probe substrates. Additionally, 19 potential products of enzymatic oxidation were generated via two additional synthetic steps for use as product standards in future studies.

CYP108N12 initiates p-cymene biodegradation in Rhodococcus globerulus.

Rhodococcus globerulus (R. globerulus) isolated from soil beneath Eucalyptus sp. was found to live on the monoterpenes 1,8-cineole, p-cymene and (R)- and (S)-limonene as sole sources of carbon and energy. Previous metabolic studies revealed that R. globerulus is capable of living on 1,8-cineole, the main monoterpene component of eucalyptus essential oil through the activity of cytochrome P450cin (CYP176A1) [1]. Genomic sequencing of R. globerulus revealed a novel putative cytochrome P450 (CYP108N12) that shares 48% sequence identity with CYP108A1 (P450terp) from Pseudomonas sp., an α-terpineol hydroxylase. Given the sequence similarity between CYP108N12 and P450terp, it was hypothesised that CYP108N12 may be responsible for initiating the biodegradation of a monoterpene structurally similar to α-terpineol such as (R)-limonene, (S)-limonene or p-cymene. Encoded within the operon containing CYP108N12 were two putative bacterial P450 redox partners and putative alcohol and aldehyde dehydrogenases, suggesting a complete catalytic system for activating these monoterpenes. Binding studies revealed that p-cymene and (R)- and (S)-limonene all bound tightly to CYP108N12 but α-terpineol did not. A catalytically active system was reconstituted using the non-native redox partner putidaredoxin and putidaredoxin reductase that act with CYP101A1 (P450cam) from Pseudomonas. This reconstituted system catalysed the hydroxylation of p-cymene to 4-isopropylbenzyl alcohol, and (R)- and (S)-limonene to (R)- and (S)-perillyl alcohol, respectively. R. globerulus was successfully grown on solely p-cymene, (R)-limonene or (S)-limonene. CYP108N12 was detected when R. globerulus was grown on p-cymene, but not either limonene enantiomer. The native function of CYP108N12 is therefore proposed to be initiation of p-cymene biodegradation by methyl oxidation and is a potentially attractive biocatalyst capable of specific benzylic and allylic hydroxylation.

To Be, or Not to Be, an Inhibitor: A Comparison of Azole Interactions with and Oxidation by a Cytochrome P450 Enzyme.

The cytochrome P450 (CYP) superfamily of heme monooxygenases is involved in a range of important chemical biotransformations across nature. Azole-containing molecules have been developed as drugs that bind to the heme center of these enzymes, inhibiting their function. The optical spectrum of CYP enzymes after the addition of these inhibitors is used to assess how the molecules bind. Here we use the bacterial CYP199A4 enzyme, from Rhodopseudomonas palustris HaA2, to compare how imidazolyl and triazolyl inhibitors bind to ferric and ferrous heme. 4-(Imidazol-1-yl)benzoic acid induced a red shift in the Soret wavelength (424 nm) in the ferric enzyme along with an increase and a decrease in the intensities of the δ and α bands, respectively. 4-(1H-1,2,4-Triazol-1-yl)benzoic acid binds to CYP199A4 with a 10-fold lower affinity and induces a smaller red shift in the Soret band. The crystal structures of CYP199A4 with these two inhibitors confirmed that these differences in the optical spectra were due to coordination of the imidazolyl ligand to the ferric Fe, but the triazolyl inhibitor interacts with, rather than displaces, the ferric aqua ligand. Additional water molecules were present in the active site of 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound CYP199A4. The space required to accommodate these additional water molecules in the active site necessitates changes in the position of the hydrophobic phenylalanine 298 residue. Upon reduction of the heme, the imidazole-based inhibitor Fe-N ligation was not retained. A 5-coordinate heme was also the predominant species in 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound ferrous CYP199A4, but there was an obvious shoulder at 447 nm indicative of some degree of Fe-N coordination. Rather than inhibit CYP199A4, 4-(imidazol-1-yl)benzoic acid was a substrate and was oxidized to generate a metabolite derived from ring opening of the imidazolyl ring: 4-[[2-(formylamino)acetyl]amino]benzoic acid.

Exploring the substrate specificity of Cytochrome P450cin.

Cytochromes P450 are enzymes that catalyse the oxidation of a wide variety of compounds that range from small volatile compounds, such as monoterpenes to larger compounds like steroids. These enzymes can be modified to selectively oxidise substrates of interest, thereby making them attractive for applications in the biotechnology industry. In this study, we screened a small library of terpenes and terpenoid compounds against P450cin and two P450cin mutants, N242A and N242T, that have previously been shown to affect selectivity. Initial screening indicated that P450cin could catalyse the oxidation of most of the monoterpenes tested; however, sesquiterpenes were not substrates for this enzyme or the N242A mutant. Additionally, both P450cin mutants were found to be able to oxidise other bicyclic monoterpenes. For example, the oxidation of (R)- and (S)-camphor by N242T favoured the production of 5-endo-hydroxycamphor (65-77% of the total products, dependent on the enantiomer), which was similar to that previously observed for (R)-camphor with N242A (73%). Selectivity was also observed for both (R)- and (S)-limonene where N242A predominantly produced the cis-limonene 1,2-epoxide (80% of the products following (R)-limonene oxidation) as compared to P450cin (23% of the total products with (R)-limonene). Of the three enzymes screened, only P450cin was observed to catalyse the oxidation of the aromatic terpene p-cymene. All six possible hydroxylation products were generated from an in vivo expression system catalysing the oxidation of p-cymene and were assigned based on 1H NMR and GC-MS fragmentation patterns. Overall, these results have provided the foundation for pursuing new P450cin mutants that can selectively oxidise various monoterpenes for biocatalytic applications.