2007LewisDGphd.pdf (5.92 MB)
Characterization of Mycobacterium sp. P450 Systems
thesisposted on 2008-08-11, 14:18 authored by David Geraint Lewis
This work reports detailed structural and mechanistic characterisation of three cytochrome P450 (CYP) enzymes from diverse mycobacteria. For Mycobacterium tuberculosis (Mtb) CYP121, a high resolution atomic structure revealed mutagenesis targets to explore structure/function relationships. Several mutants were generated and structurally characterised. The R386L variant disrupted heme coordination environment, producing an enzyme with shifted Soret absorption features and predominantly high-spin ferric heme iron. EPR and resonance Raman spectra confirmed the spin-state conversion. The P346L variant resulted in altered heme conformation due to perturbed interactions with the heme pyrrole D ring. Alterations to heme ligation state were observed from EPR studies. Ligand binding and thermodynamic studies were done on CYP121 mutants. Studies of the action of various azole based drugs (which bind tightly to CYP121 heme iron) showed that azoles had potent inhibitory effects on Mtb growth. Mycobacterium ulcerans, the aetiological agent of Buruli ulcer, produces a human toxin (mycolactone) and encodes CYP140A2 on a plasmid, with the CYP140A2 gene adjacent to other mycolactone biosynthetic genes. Purified oxidised CYP140A2 has a Soret absorption band at 418 nm and α/β bands at 567/534 nm, respectively. The Fe(II)CO complex conforms to typical P450 properties, with a characteristic Soret shift to 449 nm. Resonance Raman and EPR spectra confirm the protein to be low-spin and with a typical cysteinate- and water-ligated b-type heme iron. Azole drugs were shown to bind CYP140A2 tightly, and to be potential therapeutics. CYP140A2 catalysed hydroxylation of mycolactone precursor molecules, consistent with its proposed role in synthesis of the toxic polyketide. Mycobacterium sp HE5 CYP151A2 was shown to have a role in metabolism of secondary amines, such as morpholine. CYP151A2 was expressed and purified, and spectroscopic analyses were consistent with cysteinate- and aqua-ligated heme iron. Dithionite reduction of CYP151A2 induced non-standard spectral changes, with a blue-shifted Soret band indicative of a cysteine thiolate-to-thiol switch in heme ligation. Thiolate coordination was restored on oxidation of CYP151A2, indicating that the thiol is readily deprotonated to thiolate in the ferric form. An 419 of 186 mM-1 cm-1 was determined for oxidised CYP151A2 and (at high concentration) morpholine substrate was shown to coordinate heme iron as an inhibitor at high concentrations.
Date of award2007-01-01
Awarding institutionUniversity of Leicester