AbstractsBiology & Animal Science

Characterisation of Novel Cytochrome P450-fusion enzymes

by Dominika Luciakova

Institution: University of Manchester
Year: 2015
Keywords: Cytochrome P450, fusion enzymes, CPR, redox partner proteins, C.metallidurans; alcohol dehydrogenase
Record ID: 1399280
Full text PDF: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:244802


Characterisation of Novel Cytochrome P450-fusion enzymesSeptember 2014This study focuses on the characterisation of three novel cytochrome P450-partner (P450-fusion) enzymes of unknown structure and function. Despite several well-established P450 functions, new structures of P450s are published frequently, with the P450-redox partner fusion systems being among the most discussed, due to their enhanced activity and biotechnological potential. Other, more intriguing, P450-fusions involve partners with functions distinct from electron transfer. Understanding why evolution drove the ‘partner’ proteins to evolve into a single unit is often unclear, but provides an important challenge for the understanding of the breadth of biochemical reactions mediated by P450s.The first P450-fusion analysed (Chapter 3) is CYP116B1 from a soil bacterium, Cupriavidus metallidurans, that displays important environmental implications. The enzyme was characterised as a functional fusion, composed of three domains: a P450 from the CYP116B family, and a phthalate dioxygenase reductase (PDOR)-like reductase binding FMN and a 2Fe-2S cluster. CYP116B1 is a stable, cytosolic enzyme but can undergo FMN cofactor loss. Studies included redox potentiometry of the intact fusion and its individual domains using spectro-electrochemical and EPR methods to enable the determination of midpoint redox potentials for individual cofactors. The CYP116B1 EPR signature was shown to be typical of P450s, and changed upon binding heme-coordinating inhibitors of the azole class. Extensive compound library screening did not reveal a substrate-like physiological “hit”. However, catalytic activity was detected towards selected thiocarbamate herbicides. GC-MS data revealed the enzymatic mechanism of herbicide degradation.The second system studied (Chapter 4) is P450-CAD, an atypical fusion of an uncharacterised soluble P450 and a cinnamyl alcohol dehydrogenase (ADH) module from Streptomyces ghanaensis; a member of the major antibiotic producing genus of bacteria. The CAD module appears unlikely to be a redox partner, but instead possibly mediates substrate/product exchange with the P450. The intact fusion was shown to aggregate during extraction. Genetic dissection of domains revealed that this was due to the highly insoluble ADH moiety. The heme domain (HD) was soluble and was characterised extensively. The enzyme displays an unusual spectrum when in the FeII-CO complex (Amax = 445 nm). The P450-CAD HD catalytic activity is supported by heterologous redox partners (E. coli flavodoxin reductase [FldR] and flavodoxin [FldA], and spinach ferredoxin reductase [FdR] and ferredoxin [Fdx]). The CAD-HD binds fatty acid substrates of carbon chain length C8-14, with the highest affinity for 12-methylmyristic acid (12M14C acid), the C12 lauric acid, its aldehyde and alcohol, indicating that the terminal methyl group is important for binding to the enzyme. Unusually, the CAD-HD also binds a range of detergent compounds. Further analysis included SEC-MALLS, thermostability and…