Structure and characterisation of the cytochrome P450 redox system from M. tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of TB, has re-emerged as a global threat to human health. The Mtb genome sequence revealed 20 genes encoding cytochrome P450 enzymes (P450s), along with multiple genes for accessory redox partners. This large P450 number was unprecedented for prokaryotes and only recent sequencing projects have revealed other actinomycetes to have similarly large P450 complements. The data suggest important physiological roles for P450s in Mtb. Mycobacteria possess novel biosynthetic pathways and, given the complex composition of their envelope, it might be anticipated that many of the Mtb P450s have roles in synthesis and interconversion of membrane lipids Several P450 (CYP) and redox partner genes (a ferredoxin and two ferredoxin reductases) were cloned by PCR. CYP121 is the major P450 expressed in Mtb. Its expression is altered by various cellular stresses. CYP121 binds anti-fungal imidazoles extremely tightly and these drugs were shown to be potent growth inhibitors in mycobacteria and streptomyces spp. The CYP121 structure was solved to atomic resolution, revealing novel features of this cytochrome P450. The high resolution structure provides an insight into P450 oxygen binding, proton relay channels involving amino acids, and other important aspects of P450 catalysis. The adrenodoxin reductase homologue FprA is the favoured candidate for the ferredoxin reductase component of a Mtb P450 redox system. Kinetic characterization of the flavoprotein FprA reveals intriguing features consistent with the presence of distinct catalytic and regulatory sites for NADPH binding. FprA may be a model for this type of complex regulatory behaviour. The Mtb ferredoxin Fd-1 was shown to mediate electron transfer between reduced FprA and CYP121, reconstituting an active Mtb redox system. The electron transfer reaction was slow, and may operate against a thermodynamic gradient in vivo. This may reflect a regulatory mechanism required for control of P450 catalytic turnover rate compatible with the slow growth rates of the pathogen.