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.

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.

Characterization of the cholesterol biosynthetic pathway in Dioscorea transversa.

Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant, Dioscorea transversa, produces only two steroidal saponins: 1ß-hydroxyprotoneogracillin and protoneogracillin. Here, we used D. transversa as a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes of D. transversa rhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ24,28 double bonds required for phytosterol biogenesis as well as Δ24,25 double bonds. The latter function is believed to initiate cholesterogenesis by reducing cycloartenol to cycloartanol. Through heterologous expression, purification, and enzymatic reconstitution, we also demonstrate that the D. transversa sterol demethylase (CYP51) effectively demethylates obtusifoliol, an intermediate of phytosterol biosynthesis and 4-desmethyl-24,25-dihydrolanosterol, a postulated downstream intermediate of cholesterol biosynthesis. In summary, we investigated specific steps of the cholesterol biosynthetic pathway, providing further insight into the downstream production of bioactive steroidal saponin metabolites.

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.

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.

Structural insights into the role of the acid-alcohol pair of residues required for dioxygen activation in cytochrome P450 enzymes.

The cytochrome P450 heme monooxygenases commonly use an acid-alcohol pair of residues, within the I-helix, to activate iron-bound dioxygen. This work aims to clarify conflicting reports on the importance of the alcohol functionality in this process. Mutants of the P450, CYP199A4 (CYP199A4D251N and CYP199A4T252A), were prepared, characterised and their crystal structures were solved. The acid residue of CYP199A4 is not part of a salt bridge network, a key feature of paradigmatic model system P450cam. Instead, there is a direct proton delivery network, via a chain of water molecules, extending to the surface. Nevertheless, CYP199A4D251N dramatically reduced the activity of the enzyme consistent with a role in proton delivery. CYP199A4T252A decreased the coupling efficiency of the enzyme with a concomitant increase in the hydrogen peroxide uncoupling pathway. However, the effect of this mutation was much less pronounced than reported with P450cam. Its crystal structures revealed fewer changes at the I-helix, compared to the P450cam system. The structural changes observed within the I-helix of P450cam during oxygen activation do not seem to be required in this P450. These differences are due to the presence of a second threonine residue at position 253, which is absent in P450cam. This threonine forms part of the hydrogen bonding network, resulting in subtle structural changes and is also present across the majority of the P450 superfamily. Overall, the results suggest that while the acid-alcohol pair is important for dioxygen activation this process and the method of proton delivery can differ across P450s.Graphic abstract.

Biophysical Techniques for Distinguishing Ligand Binding Modes in Cytochrome P450 Monooxygenases.

The cytochrome P450 superfamily of heme monooxygenases catalyzes important chemical reactions across nature. The changes in the optical spectra of these enzymes, induced by the addition of substrates or inhibitors, are critical for assessing how these molecules bind to the P450, enhancing or inhibiting the catalytic cycle. Here we use the bacterial CYP199A4 enzyme (Uniprot entry Q2IUO2), from Rhodopseudomonas palustris HaA2, and a range of substituted benzoic acids to investigate different binding modes. 4-Methoxybenzoic acid elicits an archetypal type I spectral response due to a ≥95% switch from the low- to high-spin state with concomitant dissociation of the sixth aqua ligand. 4-(Pyridin-3-yl)- and 4-(pyridin-2-yl)benzoic acid induced different type II ultraviolet-visible (UV-vis) spectral responses in CYP199A4. The former induced a greater red shift in the Soret wavelength (424 nm vs 422 nm) along with a larger overall absorbance change and other differences in the α-, ß-, and δ-bands. There were also variations in the ferrous UV-vis spectra of these two substrate-bound forms with a spectrum indicative of Fe-N bond formation with 4-(pyridin-3-yl)benzoic acid. The crystal structures of CYP199A4, with the pyridinyl compounds bound, revealed that while the nitrogen of 4-(pyridin-3-yl)benzoic acid is coordinated to the heme, with 4-(pyridin-2-yl)benzoic acid an aqua ligand remains. Continuous wave and pulse electron paramagnetic resonance data in frozen solution revealed that the substrates are bound in the active site in a form consistent with the crystal structures. The redox potential of each CYP199A4-substrate combination was measured, allowing correlation among binding modes, spectroscopic properties, and the observed biochemical activity.

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.

The cubane paradigm in bioactive molecule discovery: further scope, limitations and the cyclooctatetraene complement.

The cubane phenyl ring bioisostere paradigm was further explored in an extensive study covering a wide range of pharmaceutical and agrochemical templates, which included antibiotics (cefaclor, penicillin G) and antihistamine (diphenhydramine), a smooth muscle relaxant (alverine), an anaesthetic (ketamine), an agrochemical instecticide (triflumuron), an antiparasitic (benznidazole) and an anticancer agent (tamibarotene). This investigation highlights the scope and limitations of incorporating cubane into bioactive molecule discovery, both in terms of synthetic compatibility and physical property matching. Cubane maintained bioisosterism in the case of the Chagas disease antiparasitic benznidazole, although it was less active in the case of the anticancer agent (tamibarotenne). Application of the cyclooctatetraene (COT) (bio)motif complement was found to optimize benznidazole relative to the benzene parent, and augmented anticancer activity relative to the cubane analogue in the case of tamibarotene. Like all bioisosteres, scaffolds and biomotifs, however, there are limitations (e.g. synthetic implementation), and these have been specifically highlighted herein using failed examples. A summary of all templates prepared to date by our group that were biologically evaluated strongly supports the concept that cubane is a valuable tool in bioactive molecule discovery and COT is a viable complement.

Significance of Protein-Substrate Hydrogen Bonding for the Selectivity of P450-Catalysed Oxidations.

P450cin and P450cam are bacterial cytochromes P450 that specifically hydroxylate bicyclic monoterpenes. Protein-substrate H bonding has been previously proposed as crucial in the selectivity of P450cin oxidations, but not as essential for P450cam . To examine the difference in importance of H bonds in these two model P450s, the P450-catalysed oxidation products from thiocamphor were compared. Surprisingly, both P450s oxidised thiocamphor predominantly to the corresponding S-oxides, in contrast to previous reports, and this is the first report of P450-catalysed sulfine generation from a thioketone. Additionally, the result emphasised the importance of the protein-substrate H bond to selectivity in both P450cin and P450cam . The H bonding in P450cam was re-examined using camphane, another substrate for which the protein-substrate H bond is absent. The results indicated that both H bonding and hydrophobic interactions between substrate and protein play a role in selectivity. Interestingly, the protein-substrate H bond was not a factor in substrate affinity for the enzyme.

Selective hydroxylation of 1,8- and 1,4-cineole using bacterial P450 variants.

This study has evaluated the use of the P450 metalloenzymes CYP176A1, CYP101A1 and CYP102A1, together with engineered protein variants of CYP101A1 and CYP102A1, to alter the regioselectivity of 1,8- and 1,4-cineole hydroxylation. CYP176A1 was less selective for 1,4-cineole oxidation when compared to its preferred substrate, 1,8-cineole. The CYP102A1 variants significantly improved the activity over the WT enzyme for oxidation of 1,4- and 1,8-cineole. The CYP102A1 R47L/Y51F/A74G/F87V/L188Q mutant generated predominantly (1S)-6α-hydroxy-1,8-cineole (78% e.e.) from 1,8-cineole. Oxidation of 1,4-cineole by the CYP102A1 R47L/Y51F/F87A/I401P variant generated the 3α product in >90% yield. WT CYP101A1 formed a mixture metabolites with 1,8-cineole and very little product was generated with 1,4-cineole. In contrast the F87W/Y96F/L244A/V247L and F87W/Y96F/L244A variants of CYP101A1 favoured formation of 5α-hydroxy-1,8-cineole (>88%, 1S 86% e.e.) while the F87V/Y96F/L244A variant generated (1S)-6α-hydroxy-1,8-cineole in excess (90% regioselective, >99% e.e.). The CYP101A1 F87W/Y96F/L244A/V247L and F87W/Y96F/L244A mutants improved the oxidation of 1,4-cineole generating an excess of the 3α metabolite (1S > 99% e.e. with the latter). The CYP101A1 F87L/Y96F variant also improved the oxidation of this substrate but shifted the site of oxidation to the isopropyl group, (8-hydroxy-1,4-cineole). When this 8-hydroxy metabolite was generated in significant quantities desaturation of C8C9 to the corresponding alkene was also detected.

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism.

Covering: 2000 up to 2018 The cytochromes P450 (P450s) are a superfamily of heme-containing monooxygenases that perform diverse catalytic roles in many species, including bacteria. The P450 superfamily is widely known for the hydroxylation of unactivated C-H bonds, but the diversity of reactions that P450s can perform vastly exceeds this undoubtedly impressive chemical transformation. Within bacteria, P450s play important roles in many biosynthetic and biodegradative processes that span a wide range of secondary metabolite pathways and present diverse chemical transformations. In this review, we aim to provide an overview of the range of chemical transformations that P450 enzymes can catalyse within bacterial secondary metabolism, with the intention to provide an important resource to aid in understanding of the potential roles of P450 enzymes within newly identified bacterial biosynthetic pathways.

Validating Eaton's Hypothesis: Cubane as a Benzene Bioisostere.

Pharmaceutical and agrochemical discovery programs are under considerable pressure to meet increasing global demand and thus require constant innovation. Classical hydrocarbon scaffolds have long assisted in bringing new molecules to the market place, but an obvious omission is that of the Platonic solid cubane. Eaton, however, suggested that this molecule has the potential to act as a benzene bioisostere. Herein, we report the validation of Eaton's hypothesis with cubane derivatives of five molecules that are used clinically or as agrochemicals. Two cubane analogues showed increased bioactivity compared to their benzene counterparts whereas two further analogues displayed equal bioactivity, and the fifth one demonstrated only partial efficacy. Ramifications from this study are best realized by reflecting on the number of bioactive molecules that contain a benzene ring. Substitution with the cubane scaffold where possible could revitalize these systems, and thus expedite much needed lead candidate identification.

Direct Observation of an Oxepin from a Bacterial Cytochrome P450-Catalyzed Oxidation.

The cytochromes P450 are hemoproteins that catalyze a range of oxidative C-H functionalization reactions, including aliphatic and aromatic hydroxylation. These transformations are important in a range of biological contexts, including biosynthesis and xenobiotic biodegradation. Much work has been carried out on the mechanism of aliphatic hydroxylation, implicating hydrogen atom abstraction, but aromatic hydroxylation is postulated to proceed differently. One mechanism invokes as the key intermediate an arene oxide (and/or its oxepin tautomer). Conclusive isolation of this intermediate has remained elusive and, currently, direct formation of phenols from a Meisenheimer intermediate is believed to be favored. We report here the identification of a P450 [P450cam (CYP101A1) and P450cin (CYP176A1)]-generated arene oxide as a product of in vitro oxidation of tert-butylbenzene. Computations (CBS-QB3) predict that the arene oxide and oxepin have similar stabilities to other arene oxides/oxepins implicated (but not detected) in P450-mediated transformations, suggesting that arene oxides can be unstable terminal products of P450-catalyzed aromatic oxidation that can explain the origin of some observed metabolites.

Cytochrome P450cin (CYP176A1).

Cytochrome P450cin (P450cin) (CYP176A1) is a bacterial P450 enzyme that catalyses the enantiospecific hydroxylation of 1,8-cineole to (1R)-6ß-hydroxycineole when reconstituted with its natural reduction-oxidation (redox) partner cindoxin, E. coli flavodoxin reductase, and NADPH as a source of electrons. This catalytic system has become a useful tool in the study of P450s as not only can large quantities of P450cin be prepared and rates of oxidation up to 1,500 min(-1) achieved, but it also displays a number of unusual characteristics. These include an asparagine residue in P450cin that has been found in place of the usual conserved threonine residue observed in most P450s. In general, this conserved threonine controls oxygen activation to create the potent ferryl (Fe(IV=O) porphyrin cation radical required for substrate oxidation. Another atypical characteristic of P450cin is that it utilises an FMN-containing redoxin (cindoxin) rather than a ferridoxin as is usually observed with other bacterial P450s (e.g. P450cam). This chapter will review what is currently known about P450cin and how this enzyme has provided a greater understanding of P450s in general.

Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB.

Pseudomonas putida GPo1 alkane hydroxylase (AlkB) is an integral membrane protein that catalyses the hydroxylation of medium-chain alkanes (C3-C12). 1-Octyne irreversibly inhibits this non-haem di-iron mono-oxygenase under turnover conditions, suggesting that it acts as a mechanism-based inactivator. Upon binding to the active site, 1-octyne is postulated to be oxidized to an oxirene that rapidly rearranges to a reactive ketene which covalently acylates nearby residues, resulting in enzyme inactivation. In analysis of inactivated AlkB by LC-MS/MS, several residues exhibited a mass increase of 126.1 Da, corresponding to the octanoyl moiety derived from oxidative activation of 1-octyne. Mutagenesis studies of conserved acylated residues showed that Lys18 plays a critical role in enzyme function, as a single-point mutation of Lys18 to alanine (K18A) completely abolished enzymatic activity. Finally, we present a computational 3D model structure of the transmembrane domain of AlkB, which revealed the overall packing arrangement of the transmembrane helices within the lipid bilayer and the location of the active site mapped by the 1-octyne modifications.

Cytochrome P450(cin) (CYP176A1) D241N: investigating the role of the conserved acid in the active site of cytochrome P450s.

P450(cin) (CYP176A) is a rare bacterial P450 in that contains an asparagine (Asn242) instead of the conserved threonine that almost all other P450s possess that directs oxygen activation by the heme prosthetic group. However, P450(cin) does have the neighbouring, conserved acid (Asp241) that is thought to be involved indirectly in the protonation of the dioxygen and affect the lifetime of the ferric-peroxo species produced during oxygen activation. In this study, the P450(cin) D241N mutant has been produced and found to be analogous to the P450(cam) D251N mutant. P450(cin) catalyses the hydroxylation of cineole to give only (1R)-6ß-hydroxycineole and is well coupled (NADPH consumed: product produced). The P450(cin) D241N mutant also hydroxylated cineole to produce only (1R)-6ß-hydroxycineole, was moderately well coupled (31±3%) but a significant reduction in the rate of the reaction (2% as compared to wild type) was observed. Catalytic oxidation of a variety of substrates by D241N P450(cin) were used to examine if typical reactions ascribed to the ferric-peroxo species increased as this intermediate is known to be more persistent in the P450(cam) D251N mutant. However, little change was observed in the product profiles of each of these substrates between wild type and mutant enzymes and no products consistent with chemistry of the ferric-peroxo species were observed to increase.

Cytochrome P450 is present in both ferrous and ferric forms in the resting state within intact Escherichia coli and hepatocytes.

Cytochrome P450 enzymes (P450s) are exceptionally versatile monooxygenases, mediating hydroxylations of unactivated C-H bonds, epoxidations, dealkylations, and N- and S-oxidations as well as other less common reactions. In the conventional view of the catalytic cycle, based upon studies of P450s in vitro, substrate binding to the Fe(III) resting state facilitates the first 1-electron reduction of the heme. However, the resting state of P450s in vivo has not been examined. In the present study, whole cell difference spectroscopy of bacterial (CYP101A1 and CYP176A1, i.e. P450cam and P450cin) and mammalian (CYP1A2, CYP2C9, CYP2A6, CYP2C19, and CYP3A4) P450s expressed within intact Escherichia coli revealed that both Fe(III) and Fe(II) forms of the enzyme are present in the absence of substrates. The relevance of this finding was supported by similar observations of Fe(II) P450 heme in intact rat hepatocytes. Electron paramagnetic resonance (EPR) spectroscopy of the bacterial forms in intact cells showed that a proportion of the P450 in cells was in an EPR-silent form in the native state consistent with the presence of Fe(II) P450. Coexpression of suitable cognate electron donors increased the degree of endogenous reduction to over 80%. A significant proportion of intracellular P450 remained in the Fe(II) form after vigorous aeration of cells. The addition of substrates increased the proportion of Fe(II) heme, suggesting a kinetic gate to heme reduction in the absence of substrate. In summary, these observations suggest that the resting state of P450s should be regarded as a mixture of Fe(III) and Fe(II) forms in both aerobic and oxygen-limited conditions.

Oxygen activation by P450(cin): Protein and substrate mutagenesis.

A conserved threonine found in the majority of cytochromes P450 (P450s) has been implicated in the activation of dioxygen during the catalytic cycle. P450(cin) (CYP176A) has been found to be an exception to this paradigm, where the conserved threonine has been replaced with an asparagine. Prior studies with a P450(cin) N242A mutant established that the Asn-242 was not a functional replacement for the conserved threonine but was essential for the regio- and stereocontrol of the oxidation of cineole. To explore further how P450(cin) controls the activation of the dioxygen in the absence of the conserved threonine, two concurrent lines of investigation were followed. Modification of P450(cin) indicated that the Thr-243 was not involved in controlling the protonation of the hydroperoxy species. In addition, the N242T mutant did not enhance the rate and/or efficiency of catalytic turnover of cineole by P450(cin). In parallel experiments, the substrate cineole was modified by removing the ethereal oxygen to produce camphane or 2,2-dimethylbicyclo[2.2.2]octane (cinane). An analogous experiment with P450(EryF) showed that a hydroxyl group on the substrate was vital, and in its absence catalytic turnover was effectively abolished. Catalytic turnover of P450(cin) with either of these alternative substrates (camphane or cinane) revealed that in the absence of the ethereal oxygen there was still a significant amount of coupling of the NADPH-reducing equivalents to the formation of oxidised product. Again the substrate itself was not found to be important in controlling oxygen activation, in contrast to P450(EryF), but was shown to be essential for regio- and stereoselective substrate oxidation. Thus, it still remains unclear how dioxygen activation in the catalytic turnover of cineole by P450(cin) is controlled.