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.

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.

Thermo-alkaline Treatment as a Practical Degradation Strategy To Reduce Indospicine Contamination in Camel Meat.

Ingestion of indospicine-contaminated camel and horse meat has caused fatal liver injury to dogs in Australia, and it is currently not known if such contaminated meat may pose a human health risk upon dietary exposure. To date, indospicine-related research has tended to focus on analytical aspects, with little information on post-harvest management of indospicine-contaminated meat. In this study, indospicine degradation was investigated in both aqueous solution and also contaminated meat, under a range of conditions. Aqueous solutions of indospicine and indospicine-contaminated camel meat were microwaved (180 °C) or autoclaved (121 °C) with the addition of food-grade additives [0.05% (v/v) acetic acid or 0.05% (w/v) sodium bicarbonate] for 0, 15, 30, and 60 min. An aqueous sodium bicarbonate solution demonstrated the greatest efficacy in degrading indospicine, with complete degradation after 15 min of heating in a microwave or autoclave; concomitant formation of indospicine degradation products, namely, 2-aminopimelamic and 2-aminopimelic acids, was observed. Similar treatment of indospicine-contaminated camel meat with aqueous sodium bicarbonate resulted in 50% degradation after 15 min of heating in an autoclave and 100% degradation after 15 min of heating in a microwave. The results suggest that thermo-alkaline aqueous treatment has potential as a pragmatic post-harvest handling technique in reducing indospicine levels in indospicine-contaminated meat.

Synthesis of l-indospicine, [5,5,6-(2)H3]-l-indospicine and l-norindospicine.

Indospicine is a non-proteogenic amino acid that accumulates as the free amino acid in livestock grazing Indigofera plant species and causes both reproductive losses and hepatotoxic effects. An efficient synthetic route to l-indospicine from l-homoserine lactone is described. The methodology is applicable for the synthesis of both deuterium labelled isotopomers and structural analogues for utilisation in biological studies. The key steps are a zinc mediated Barbier reaction with acrylonitrile and a Pinner reaction that together introduce the target amidine moiety.

Cholesterol Analogs with Degradation-resistant Alkyl Side Chains Are Effective Mycobacterium tuberculosis Growth Inhibitors.

Cholest-4-en-3-one, whether added exogenously or generated intracellularly from cholesterol, inhibits the growth ofMycobacterium tuberculosiswhen CYP125A1 and CYP142A1, the cytochrome P450 enzymes that initiate degradation of the sterol side chain, are disabled. Here we demonstrate that a 16-hydroxy derivative of cholesterol, which was previously reported to inhibit growth ofM. tuberculosis, acts by preventing the oxidation of the sterol side chain even in the presence of the relevant cytochrome P450 enzymes. The finding that (25R)-cholest-5-en-3ß,16ß,26-triol (1) (and its 3-keto metabolite) inhibit growth suggests that cholesterol analogs with non-degradable side chains represent a novel class of anti-mycobacterial agents. In accord with this, two cholesterol analogs with truncated, fluorinated side chains have been synthesized and shown to similarly block the growth in culture ofM. tuberculosis.