Biochemical and structural characterisation of the peroxygenase P450 KR

  • Jimmy Tan

Student thesis: Master of Philosophy


Several research groups have reported biosynthesis of alkanes and alkenes by enzymes such as the aldehyde deformylating oxygenase (ADO) and OleTJE (an alkene producing P450 enzyme). This thesis reports a novel cytochrome P450 peroxygenase from the bacterium Kocuria rhizophila (termed P450 KR) which is shown to convert saturated fatty acids to the corresponding n-1 terminal alkenes by oxidative decarboxylation.UV-visible spectroscopic studies showed that P450 KR has a resting state, low-spin haem Soret band at 424 nm, which is a longer wavelength than most other P450s, and suggests an unusual coordination state of the haem. P450 KR binds short- to medium-chain fatty acids ranging from caprylic acid (C8:0) to palmitic acid (16:0), and converts these substrates to alkenes and hydroxylated fatty acids. Short-chain fatty acids bind more weakly compared to the long-chain lipids, with dissociation constants (Kd values) ranging from millimolar to micromolar. Notwithstanding weak binding observed for short-chain fatty acids, their relatively high aqueous solubility enables many to quite effectively saturate the P450 KR active site and to produce substantial high-spin ferric haem content. These findings are rather different to those seen in OleTJE. Electron paramagnetic resonance (EPR) spectroscopy reveals complex, heterogeneous P450 KR spectra that suggest that the haem iron has multiple interaction partner molecules. Crystallographic data present an interesting structure for P450 KR, existing as a dimer formed through zipper-like interactions between the N-terminal helices of both the monomers. A disulphide bridge is observed within each of the monomers of the P450 KR dimer, which is extremely unusual in a P450. These disulphide bridges apparently "lock" the extended N-terminal helix in a fixed position which promotes its interaction with the opposing helix to facilitate dimerisation. In light of these unusual structural features of P450 KR, multi-angle laser light scattering (MALLS) studies were done to examine the aggregation state of P450 KR. MALLS data revealed that P450 KR exists in a monomer/dimer equilibrium with a dimerization constant (Kd) of ~6 M. Analytical ultracentrifugation (AUC) analysis of P450 KR also reveals a monomer/dimer equilibrium. Bead modelling of the P450 KR structure matches well with the AUC data, confirming that monomer and dimer forms co-exist in solution. Fatty acid substrate binding titrations and turnover studies using concentrated P450 KR confirms that the dimer form of P450 KR is catalytically functional in addition to the monomer.The data presented in this thesis confirm that P450 KR is capable of generating terminal alkenes using fatty acids as feedstock, opening up opportunities for its application in the biofuel industry. Furthermore, P450 KR exhibits an unusual monomer-dimer equilibrium in solution, which is rarely reported for P450s. The occurrence of disulphide bridges within the P450 KR monomers is also rare in P450s, but may be an important evolutionary adaptation in P450 KR which enables it to dimerise, possibly to enhance enzyme stability. Another unusual property of P450 KR is its haem Soret maximum at 424 nm in its resting ferric state, a feature that suggests the haem has a strong axial ligand interaction network. Data presented here illustrate an unusual P450 enzyme that has potentially important applications in biofuel production.
Date of Award31 Dec 2016
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorAndrew Munro (Supervisor) & David Leys (Supervisor)


  • sedimentation velocity
  • sedimentation equilibrium
  • EPR
  • X-ray crystallography
  • alkene
  • drop-in biofuel
  • alkane
  • fatty acid
  • chromatography
  • biofuel
  • headspace GC-MS
  • electron paramagnetic resonance
  • P450
  • Kocuria rhizophila
  • P450 KR
  • KR
  • cytochrome P450
  • substrate binding titration
  • KR analytical ultracentrifugation
  • AUC
  • GC-MS
  • multi-angle laser light scattering

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