Linking cytochrome P450 enzymes from Mycobacterium tuberculosis to their cognate ferredoxin partners

Abstract

Mycobacterium tuberculosis (Mtb) codes for 20 cytochrome P450 enzymes (CYPs), considered potential drug-targets due to their essential roles in bacterial viability and host infection. Catalytic activity of mycobacterial CYPs is dependent on electron transfer from a NAD (P)H-ferredoxin-reductase (FNR) and a ferredoxin (Fd). Two FNRs (FdrA and FprA) and five ferredoxins (Fdx, FdxA, FdxC, FdxD, and Rv1786) have been found in the Mtb genome. However, as of yet, the cognate redox partnerships have not been fully established. This is confounded by the fact that heterologous redox partners are routinely used to reconstitute Mtb CYP metabolism. To this end, this study aimed to biochemically characterize and identify cognate redox partnerships for Mtb CYPs. Interestingly, all combinations of FNRs and ferredoxins were active in the reduction of oxidized cytochrome c, but steady-state kinetic assays revealed FdxD as the most efficient redox partner for FdrA, whereas Fdx coupled preferably with FprA. CYP121A1, CYP124A1, CYP125A1, and CYP142A1 metabolism with the cognate redox partners was reconstituted in vitro showing an unanticipated selectivity in the requirement for electron transfer partnership, which did not necessarily correlate with proximity in the genome. This is the first description of microbial P450 metabolism in which multiple ferredoxins are functionally linked to multiple CYPs.

Keywords: Cytochrome P450; Ferredoxin; Mycobacterium tuberculosis; NAD (P) H ferredoxin reductase; Redox partners.

Fig. 1

Visual representation of the location of FdrA (Rv0688), FprA (Rv3106), Fdx (Rv0763c), FdxA (Rv2007c), FdxC (Rv1177), FdxD (Rv3503c), FdxE (Rv1786), CYP121A1 (Rv2276), CYP123A1 (Rv0766c), CYP124A1 (Rv2266), CYP125A1 (Rv3545c), CYP126A1 (Rv0778), CYP142A (Rv3518c), CYP143A1 (Rv1785c), and CYP144A1 (Rv1777) in the Mtb H37Rv genome. Genomic location of the depicted genes was retrieved from Institut Pasteur GenoList World-Wide

Fig. 2

Sequence alignment analysis of ferredoxins from Mtb strain H37Rv. a Sequence alignment of the [3Fe-4S] ferredoxins, namely Fdx, FdxD, and Rv1786/FdxE and FdsoyB found in S. griseus. Cysteines corresponding to the Cys-X₂-X-X₂-Cys-X_n-Cys-Pro iron-sulfur cluster-binding motif are highlighted with a black square. b Sequence alignment of the [3Fe-4S] and [4Fe-4S] ferredoxins from Mtb strain H37Rv, namely FdxA and FdxC, and FdxA from M. smegmatis (MsFd). Cysteines linked to the [3Fe-4S] Cys-X₂-X-X₂-Cys-X_n-Cys-Pro iron-sulfur cluster-binding motif are highlighted with a black square whereas the ones involved in the [4Fe-4S] iron-sulfur cluster-binding motif are highlighted with a dashed black square. The characteristic second Cys found in the Cys-X₂-X-X₂-Cys cluster-binding motif in [4Fe-4S] ferredoxins is highlighted with a red square

Fig. 3

UV/Vis absorption for oxidized forms of purified recombinant proteins. a FdrA. b FprA. c Fdx. d FdxA. e FdxD. f Rv1786. FdrA and FprA showed the characteristic flavin absorbance at between 300 and 500 nm, with bands centered at 380 and 452 nm and shoulders at 422 and 473 nm. Fdx, FdxD, and Rv1786/FdxE showed a broad band at 400 nm with peak at 412 nm, characteristic of [3Fe-4S] Fds whereas FdxA displayed a peak at 425 nm, characteristic of [4Fe-4S] ferredoxins

Fig. 4

cYY conversion catalyzed by CYP121A1 supported by cognate redox partners. a Extracted ion chromatograms (m/z = 325.11 [M + H⁺]) of incubations with cYY (100 μM, final concentration) and CYP121 (5 μM). Incubations were performed in a 1:5:10 M ratio. b Mycocyclosin formation by CYP121A1 (5 μM) supported by cognate redox partners. Incubations were conducted in 1:5:10 M ratio. Error bars represent the variability of duplicates

Fig. 5

Oxidative metabolism of cholesterol by recombinant Mtb CYPs reconstituted with cognate redox partners. a CYP124A1, b CYP125A1, and c CYP142A1, using different CYP-NAD(P)H FNR:Fd molar ratios. The catalytic system CYP142A1:FdrA:FdxD yielded the higher 27-hydroxycholesterol formation, and therefore, it was at 100% for comparison reasons. Error bars represent the variability of duplicates

Fig. 6

Schematic representation of redox pairs selectivity in Mtb CYPs. a Lines indicate identified in vitro specific protein interactions yielding catalytically active Mtb CYPs in this study. b In vitro specific protein interactions yielding catalytically active Mtb CYP51B1 identified by Zanno et al. (2005) and McLean et al. (2006). Preferred interactions yielding higher product formation are depicted with darker solid lines. Abbreviations: cYY cyclodipeptide cyclo (L-Tyr-L-Tyr), FF-MAS 14-demethyl-14-dehydrolanosterol