TY - JOUR
T1 - Linking cytochrome P450 enzymes from Mycobacterium tuberculosis to their cognate ferredoxin partners
AU - Ortega Ugalde, Sandra
AU - de Koning, Coen P
AU - Wallraven, Kerstin
AU - Bruyneel, Ben
AU - Vermeulen, Nico P E
AU - Grossmann, Tom N
AU - Bitter, Wilbert
AU - Commandeur, Jan N M
AU - Vos, J Chris
PY - 2018/11
Y1 - 2018/11
N2 - 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.
AB - 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.
KW - Amino Acid Sequence
KW - Cytochrome P-450 Enzyme System/metabolism
KW - Cytochrome P450
KW - Electron Transport/physiology
KW - Ferredoxin
KW - Ferredoxins/metabolism
KW - Kinetics
KW - Mycobacterium tuberculosis
KW - Mycobacterium tuberculosis/metabolism
KW - NAD (P) H ferredoxin reductase
KW - Oxidation-Reduction
KW - Oxidoreductases/metabolism
KW - Redox partners
KW - Sequence Alignment
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UR - http://www.scopus.com/inward/citedby.url?scp=85052314111&partnerID=8YFLogxK
U2 - https://doi.org/10.1007/s00253-018-9299-4
DO - https://doi.org/10.1007/s00253-018-9299-4
M3 - Article
C2 - 30136203
SN - 0175-7598
VL - 102
SP - 9231
EP - 9242
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
IS - 21
ER -