Biochemical characterisation of peroxygenase enzymes for use in the fuel industry

Abstract

The bacterial CYP152 peroxygenases are a subset of the cytochrome P450 family that have evolved to efficiently harness hydrogen peroxide as the sole oxygen and hydrogen donor during catalysis, bypassing the use of redox-partner proteins. Disocovery of P450 OleTJE (CYP152L1) isolated from Jeotgalicoccus sp. 8456 was shown to primarily functions as a fatty acid decarboxylase (C20:0 – C12:0), producing terminal alkenes. These alkenes are well suited to be implemented into the existing fuel infrastructure and provide an ideal alternative to transportation fuels, due to their ‘drop in’ compatibility. This thesis describes biochemical and structural characterisation of three novel members of the CYP152 family from Kocuria rhizophila (P450 KR, CYP152T1), Corynebacterium efficiens (P450 CE, CYP152T7) and Dietzia cinnamea (P450 DC, CYP152T8). All three orthologues described are capable of decarboxylating and/or hydroxylating saturated fatty acids (C10:0 – C18:0), with overlapping substrate specificity towards myristic acid (C14:0). While P450 KR functions primarily as a fatty acid hydroxylase, P450 CE and DC preferentially act as fatty acid decarboxylases. In contrast to the monomeric form of OleTJE, both P450 KR and P450 CE function as dimers, revealing novel dimeric interfaces in their respective crystal structures. P450 KR was crystallised and structure determined to 3.0 Å, revealing an extended N-terminal helix that protrudes from the core of the protein, and forms a “zipper-like” interaction with the adjacent monomer. These interactions appear stabilised by a disulphide linkage that contributes to protein stability. A surface disulphide linkage is also present in P450 CE in a similar position to P450 KR. However, the P450 CE structure (determined to 2.05 Å) revealed a distinct dimer interface, an additional heme molecule coordinated between two histidine residues (His186) of each monomer as seen in and stabilised by multiple hydrophobic interactions. The structure of P450 CE H186N variant (determined to 2.06 Å) lacked the extra heme group, but retained the dimer interface through the extensive hydrophobic interactions. These data provide functional and structural insights into oligomerisation, substrate-binding and product diversity of the CYP152 family.

Date of Award	31 Dec 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Munro (Supervisor) & David Leys (Supervisor)