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.

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.

Investigating the Active Oxidants Involved in Cytochrome P450 Catalyzed Sulfoxidation Reactions.

Cytochrome P450 (CYP) heme-thiolate monooxygenases catalyze the hydroxylation of the C-H bonds of organic molecules. This reaction is initiated by a ferryl-oxo heme radical cation (Cpd I). These enzymes can also catalyze sulfoxidation reactions and the ferric-hydroperoxy complex (Cpd 0) and the Fe(III)-H2 O2 complex have been proposed as alternative oxidants for this transformation. To investigate this, the oxidation of 4-alkylthiobenzoic acids and 4-methoxybenzoic acid by the CYP199A4 enzyme from Rhodopseudomonas palustris HaA2 was compared using both monooxygenase and peroxygenase pathways. By examining mutants at the mechanistically important, conserved acid alcohol-pair (D251N, T252A and T252E) the relative amounts of the reactive intermediates that would form in these reactions were disturbed. Substrate binding and X-ray crystal structures helped to understand changes in the activity and enabled an attempt to evaluate whether multiple oxidants can participate in these reactions. In peroxygenase reactions the T252E mutant had higher activity towards sulfoxidation than O-demethylation but in the monooxygenase reactions with the WT enzyme the activity of both reactions was similar. The peroxygenase activity of the T252A mutant was greater for sulfoxidation reactions than the WT enzyme, which is the reverse of the activity changes observed for O-demethylation. The monooxygenase activity and coupling efficiency of sulfoxidation and oxidative demethylation were reduced by similar degrees with the T252A mutant. These observations infer that while Cpd I is required for O-dealkylation, another oxidant may contribute to sulfoxidation. Based on the activity of the CYP199A4 mutants it is proposed that this is the Fe(III)-H2 O2 complex which would be more abundant in the peroxide-driven reactions.

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.

Synthesis of obtusifoliol and analogues as CYP51 substrates.

Sterol 14α-demethylases (CYP51s) are a ubiquitous superfamily of cytochrome P450 enzymes that play an essential role in sterol biosynthesis. As fungal CYP51s are the target of azole-based antifungal agents, which are facing the problem of increasing resistance, the substrate specificity of this enzyme subclass has recently garnered significant attention. Herein we report the first chemical synthesis of the final endogenous substrate of this enzyme class, obtusifoliol, in 1.3% yield across ten steps from a commercially available lanosterol mixture. Intermediates along this pathway provide a basis for further derivatisation of the sterol skeleton and future investigation into CYP51 inhibition to overcome pathogens' azole resistance.

The Stereoselective Oxidation of para-Substituted Benzenes by a Cytochrome P450 Biocatalyst.

The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates >60 nmol.(nmol-CYP)-1 .min-1 ) and with total turnover numbers of up to 20,000. Ethyl α-hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to >98 %). The crystal structure of 4-methoxybenzoic acid-bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.

Synthesis of steroidal inhibitors for Mycobacterium tuberculosis.

Oxidised derivatives of cholesterol have been shown to inhibit the growth of Mycobacterium tuberculosis (Mtb). The bacteriostatic activity of these compounds has been attributed to their inhibition of CYP125A1 and CYP142A1, two metabolically critical cytochromes P450 that initiate degradation of the sterol side chain. Here, we synthesise and characterise an extensive library of 28 cholesterol derivatives to develop a structure-activity relationship for this class of inhibitors. The candidate compounds were evaluated for MIC with virulent Mtb and in binding studies with CYP125A1 and CYP142A1 from Mtb.

Synthesis and biological profile of substituted hexahydrofuro[3,4-b]furans, a novel class of bicyclic acyl-acyl carrier protein (ACP) thioesterase inhibitors.

BACKGROUND: Weed control is a significant challenge for farmers around the globe. Of the various methods available for combatting weeds, small molecules remain the most effective and versatile technology to date. In the search for novel chemical entities with new modes of action toward herbicide-resistant weeds, we have investigated hexahydrofuro[3,4-b]furan-based acyl-acyl carrier protein (ACP) thioesterase inhibitors inspired by X-ray co-crystal structure-based modeling studies. RESULTS: By exploiting scaffold hopping concepts and molecular modeling studies we were able to identify new hexahydrofuro[3,4-b]furan-based lead structures showing promising activity in vivo against commercially important grass weeds in line with strong target affinity. CONCLUSION: The present work covers a series of novel herbicidal lead structures that possess a hexahydrofuro[3,4-b]furan scaffold as a structural key feature, carrying ortho-substituted aryloxy side chains. Based on an optimized synthetic approach a broad structure-activity relationship (SAR) study was carried out. The new compounds emerging from our modeling-inspired structural variations show good acyl-ACP thioesterase inhibition in line with promising initial herbicidal activity. Glasshouse trials showed that the hexahydrofuro[3,4-b]furans outlined herein display good control of cold and warm season grass-weed species in pre-emergence application. Remarkably, some of the novel acyl-ACP thioesterase-inhibitors also showed promising efficacy against warm season weeds that are difficult to control. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.