Cloning and expression of a putative P450 from Mycobacterium ulcerans

Mycolactones are macrocyclic polyketide toxins produced by the pathogen Mycobacterium ulcerans, the etiologic agent of the emerging human disease known as Buruli ulcer. A giant virulence plasmid in Mycobacterium ulcerans encoding giant polyketide synthases is responsible for the synthesis of the lipid toxin mycolactone. Mycobacterium ulcerans from different geographic origins produce varieties of mycolactones including mycolactone A/B, C E. Their difference is observed by thin layer chromagraph, mass spectrometry, cytopathic assays. The presence of different mycolactone correlates with plasmid variation. Australian strains lacking the hydroxyl group at C-12’ produce a mycolactone with a mass of [M + Na]+ at m/z 749, called mycolactone C. In consistency, plasmid from Australian strains has the absence of a region that includes the gene encoding a P450 hydroxylase. The product of this gene is predicted to hydroxylate the mycolactone side chain at C=12’ to produce mycolactone A/B with a mass of [M + Na]+ at m/z 765. In order to know whether the P450 in the plasmid is related with the hydroxyl group at C-12’ in mycolactone A/B, BAC 1707 sequence was blasted by NCBI ORF and one probably P450 open reading frame was found, designated P450A. The P450A sequence was cloned into expression vector PET30a from a BAC1707. The growth parameters controlling efficient expression of heme-containing P450 holoenzyme in E.coli has been tested. But most of the target protein was in the inclusion body. A new sequence, designated P450B, was identified by multi alignment which was 135 base pair shorter than the P450A. The P450B was cloned into pCWori vector and overexpressed. The P450B was purified from the soluble cytosolic fraction to electrophoretic homogeneity by affinity binding. The purified protein sequence was confirmed by mass spectrometry. The CO-reduced difference spectrum with peak at 420 nm suggests the purified protein is an inactive form of P450.

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