Cytochrome P450: taming a wild type enzyme

Protein engineering of cytochrome P450 monooxygenases (P450s) has been very successful in generating valuable non-natural activities and properties, allowing these powerful catalysts to be used for the synthesis of drug metabolites and in biosynthetic pathways for the production of precursors of artemisinin and paclitaxel. Collected experience indicates that the P450s are highly ‘evolvable’ – they are particularly robust to mutation in their active sites and readily accept new substrates and exhibit new selectivities. Their ability to adapt to new challenges upon mutation may reflect the nonpolar nature of their active sites as well as their high degree of conformational variability

Improved selectivity of an engineered multi-product terpene synthase

Mutation of the sesquiterpene synthase Cop2 was conducted with a high-throughput screen for the cyclization activity using a non-natural substrate. A mutant of Cop2 was identified that contained three amino acid substitutions. This mutant, 17H2, converted the natural substrate FPP into germacrene D-4-ol with 77% selectivity. This selectivity is in contrast to that of the parent enzyme in which germacrene D-4-ol is produced as 29% and α-cadinol is produced as 46% of the product mixture. The mutations were shown to each contribute to this selectivity, and a homology model suggested that the mutations lie near to the active site though would be unlikely to be targeted for mutation by rational methods. Kinetic comparisons show that 17H2 maintains a k_(cat)/K_M of 0.62 mM^(−1) s^(−1), which is nearly identical to that of the parent Cop2, which had a k_(cat)/K_M of 0.58 mM^(−1) s^(−1)

High-Throughput Screening for Terpene-Synthase-Cyclization Activity and Directed Evolution of a Terpene Synthase

The development of high-throughput assays can be extremely challenging, yet is essential for many applications in drug discovery and enzyme engineering

Indanoyl amino acid conjugates: Tunable elicitors of plant secondary metabolism

The unassuming nature of plants belies their viciously effective defensive strategies in the face of herbivore attack. Under the direction of, among others, octadecanoid hormones, plants respond by producing phytoalexins, bitter and toxic alkaloids, pr

Profiling of structurally labile oxylipins in plants by in situ derivatization with pentafluorobenzyl hydroxylamine

A GC-MS-based method for the simultaneous quantification of common oxylipins along with labile and highly reactive Compounds based on in situ derivatization with pentafluorobenzyl hydroxylamine to the corresponding O-2,3,4,5,6-pentafluorobenzyI oximes

Imidazopyridazine Hepatitis C Virus Polymerase Inhibitors. Structure–Activity Relationship Studies and the Discovery of a Novel, Traceless Prodrug Mechanism

By reducing the basicity of the core heterocycle in a series of HCV NS5B inhibitors, the hERG liability was reduced. The SAR was then systematically explored in order to increase solubility and enable dose escalation while retaining potency. During this exploration, a facile decarboxylation was noted and was exploited as a novel prodrug mechanism. The synthesis and characterization of these prodrugs and their utilization in chronic toxicity studies are presented