[Thesis]. Manchester, UK: The University of Manchester; 2015.
The University of ManchesterManca PovsicRational redesign of cytochrome P450 BM3 (CYP102A1)
towards industrially relevant drug metabolitesSeptember 2015Human drug metabolites
are frequently biologically active, with many implications for human health. Pharmaceutical
companies have become increasingly aware of the need to identify and test these metabolites.
The P450 BM3 enzyme from Bacillus megaterium offers substantial advantages to the
current methods of metabolite synthesis, as its soluble, catalytically self-sufficient
nature, coupled with its high catalytic activity, make P450 BM3 ideal for engineering
towards specificity for human drugs. The highly-active I401P BM3 mutant was characterized
for its reactivity towards human drugs and for the development of a human P450-like
metabolite profile. The I401P mutant exhibits binding to molecules including alkaloids,
steroids, and azole drugs, along with many other compounds. I401P binds/oxidizes human
CYP substrates, including alosetron, phenacetin, caffeine, nicotine and diclofenac.
LC-MS product identification shows that I401P BM3 forms 4OH-diclofenac, the major
human metabolite for diclofenac. I401P BM3 also produces nornicotine, the second major
human metabolite of nicotine. I401P BM3 also forms theophylline, theobromine and paraxanthine,
the three major human metabolites of caffeine. Thermostability (DSC) data show that
the I401P mutation destabilizes the BM3 heme domain in both its substrate-free and
substrate-bound forms. The I401P heme domain X-ray crystal structure reinforces previous
structural observations that the Pro401 mutation causes the BM3 protein to adopt a
high-spin, â€śsubstrate-boundâ€ť state, with a displaced heme iron axial water, producing
a â€ścatalytically primedâ€ť mutant with greater diversity in substrate selectivity. The
destabilisation of the BM3 heme domain structure due to the Pro401 mutation increases
conformational plasticity in this mutant, allowing it to function as a platform for
future mutagenesis aimed at improved binding and metabolite yield from specific drug
substrates. Further proline mutations (A330P, A330P/I401P and A82F/F87V/I401P) were
examined for increased affinity for drug substrates. The A330P mutant shows no novel
drug substrate specificity, despite its reported affinity for small molecules. The
A330P/I401P double mutant demonstrates weak binding to WT BM3 and I401P substrates,
but no synergistic effects were obtained by combining the two mutations. The double
mutant exhibits very low solvent tolerance and significant structural destabilisation.
DSC data confirms this, with the double mutant destabilising the BM3 heme domain by
up to 20 Â°C. Initial work with the A82F/F87V/I401P mutant showed increased affinity
for A82F/F87V- and I401P-type substrates, including diclofenac. LC-MS product analysis
confirms that the A82F/F87V/I401P mutant oxidises diclofenac into its major human
metabolite 4OH-diclofenac. These data indicate that human-like oxidation reactions
are feasible with BM3 mutants. In this work, proline insertion mutants were generated
that introduced novel affinity for biotechnologically relevant substrates. In particular
the I401P mutant offers an excellent platform for future biotechnological engineering.