Engineering terpenoid oxidation by cytochrome P450BM3 for fine chemical synthesis

Thesis

P450BM3 (CYP102A1) has a unique structure of having the electron-transfer reductase domain fused to the haem monooxygenase domain. The intramolecular electron transfer process makes P450BM3 catalytically self-sufficient and promotes high turnover rates. P450 enzymes activate C−H bonds via the insertion of an oxygen atom from atmospheric dioxygen and play key roles in the biosynthesis of endogenous compounds (terpenes, alkaloids and steroids).

Oxygenated terpenoid compounds have desirable properties such as aromas, flavours and medicinal effects. The initial oxygenation step in terpenoid biosynthesis from the terpene precursors is invariably catalysed by a P450 enzyme. Chapter 3 describes the engineering and application of P450BM3 variants to develop efficient, two-step chemo-enzymatic synthesis routes to high value the norisoprenoid and isoprenoid aroma compounds b-damascenone, tabanone isomers, and rose oxide by late-stage C−H activation.

The generation of diverse metabolites of common steroids via P450-catalysed direct C−H activation has important applications in the synthesis of steroidal agents for drug screening and development. Chapter 4 describes the engineering of P450BM3 for the mono- and di-hydroxylation of androstenedione (AD), dehydroepiandrosterone (DHEA) and testosterone (TST). To design altered steroid binding orientations to broaden the regioselectivity, the structures of wild type P450BM3 and the steroid aromatase CYP19A1 are overlaid to identify key regions for scanning glycine mutagenesis which greatly expanded the product range of AD, DHEA and TST. In Chapter 5, more steroidal compounds are screened to show hydroxylation at multiple sites with high regio- and stereo-selectivity for androsterone (AN), epi-androsterone (EAN), progesterone (PROG) and pregnenolone (PREG).

Authors: Chen, W

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: steroids; synthesis; norisoprenoid; P450; terpenoid; oxidation
• Subjects: P450 oxidation