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.Electronic supplementary materialThe online version of this article (10.1007/s00253-018-9299-4) contains supplementary material, which is available to authorized users.

Function, essentiality, and expression of cytochrome P450 enzymes and their cognate redox partners in Mycobacterium tuberculosis: are they drug targets?

“…Several transposon mutagenesis studies indicate that individual redox partners may not be essential under certain growth conditions, such as cholesterol rich conditions or during infection in vivo (Lamichhane et al 2003 ; Sassetti et al 2003 ; Sassetti and Rubin 2003 ; Lamichhane et al 2005 ; Rengarajan et al 2005 ; Jain et al 2007 ; Dutta et al 2010 ; Griffin et al 2011 ; Zhang et al 2012 ; DeJesus et al 2017 ). These results could be explained by redundancy of redox partners in CYP-mediated reactions previously shown not only for Mtb (Ortega Ugalde et al 2018a ) but also for other bacteria, such as M. marinum , Sorangium cellulosum So ce56 and Streptomyces peucetius DoxA (Ewen et al 2009 ; Rimal et al 2015 ; Child et al 2018 ).…”

“…This motif is consistent with a [3Fe-4S] cluster which is conserved on ferredoxins from other bacteria (Trower et al 1993 ; Duff et al 1996 ; Green et al 2003 ; Sevrioukova 2005 ; Lu et al 2017 ; Child et al 2018 ). On the other hand, FdxA and FdxC resemble FdxA from M. smegmatis ( MsFd ), a 7Fe-ferredoxin that contains both a [3Fe-4S] and a [4Fe-4S] iron-cluster combined in a single protein (Ricagno et al 2007 ; Ortega Ugalde et al 2018a ). The overall sequence identity between the [3Fe-4S] cluster-containing Mtb ferredoxins Fdx, FdxD, and FdxE is approximately 30%, whereas FprA displays approximately 40% sequence identity to the mammalian adrenoxin reductase (AdR) (Ricagno et al 2007 ; Fischer et al 2002 ; Ortega Ugalde et al 2018a ).…”

“…This reaction is a prerequisite for the entry of cholesterol into the β-oxidation pathway (Johnston et al 2010 ). However, this sequential metabolic activity was not seen when several cognate redox partners were used to reconstitute the system (Ortega Ugalde et al 2018a ). CYP125A1 is the most efficient catalyst, followed by CYP142A1 and finally CYP124A1 (Johnston et al 2010 ; Ouellet et al 2010b ).…”

“…However, some CYPs may have evolved with protein binding sites that are specific for (a) certain redox partner(s). Recently, it was shown that the activity of a small selection of Mtb CYPs was supported by several cognate redox partners in vitro (Ortega Ugalde et al 2018a ). Both Fdx and FdxD supported CYP121A1 catalytic activity with comparable efficiencies whereas FdxD and FdxE supported in vitro reconstitution of cholesterol hydroxylation by Mtb CYP124A1, CYP125A1, and CYP142A1, although with lower efficiency for FdxE.…”

“…Moreover, the FdrA-Fdx complex was capable of supporting CYP51B1 activity through the same electron transfer system (Zanno et al 2005 ). FdxA was the only ferredoxin unable to transfer electrons to any of the studied CYPs (Ortega Ugalde et al 2018a ). In addition, FdxC, which has not been successfully expressed yet, is required for optimal growth of Mtb in vitro suggesting a key role in shuttling electrons (Sassetti et al 2003 ; DeJesus et al 2017 ).…”