The preponderance of P450s in the Mycobacterium tuberculosis genome

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The genome of Mycobacterium tuberculosis (Mtb) encodes 20 different cytochrome P450 enzymes (P450s). P450s are mono-oxygenases, which are historically considered to facilitate prokaryotic usage of unusual carbon sources. However, their preponderance in Mtb strongly indicates crucial physiological functions, as does the fact that polycyclic azoles (known P450 inhibitors) have potent anti-mycobacterial effects. Recent structural and enzyme characterization data reveal novel features for at least two Mtb P450s (CYP121 and CYP51). Genome analysis, knockout studies and structural comparisons signify important roles in cell biology and pathogenesis for various P450s and redox partner enzymes in Mtb. Elucidation of structure, function and metabolic roles will be essential in targeting the P450s as an ‘Achilles heel’ in this major human pathogen.

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Revelations from the Mycobacterium tuberculosis genome

Genome sequencing has provided researchers with unprecedented insights into the metabolic repertoire, genetic context and organization of both unicellular and complex organisms. One of the most important breakthroughs was the determination of the genome sequence of the human pathogen Mycobacterium tuberculosis (Mtb) by Cole and co-workers [1]. Once thought to be virtually eradicated as a major human disease in the western world, Mtb has re-emerged in recent years as a worldwide threat to human

Cytochromes P450: general mechanism and physiological roles

The P450s (or CYPs) are haem b-containing enzymes that catalyze reductive scission of molecular oxygen at the haem iron, which leads to mono-oxygenation of a substrate bound close by and production of a molecule of water from the second oxygen atom [13]. This requires the delivery of two electrons from NAD(P)H by one or more redox partners and the delivery of two protons, usually from bulk solvent, mediated by active site amino acid side chains [14] (Figure 1). Reactive intermediates in the

The Mtb P450 complement: a potential role for CYP51?

There were surprises in store when the P450 complement, or ‘CYPome’, of Mtb was examined. The first prokaryotic example of the evolutionarily ancient sterol demethylase CYP51 family was immediately obvious [11]. CYP51 from Mtb was suggested to be a progenitor for the entire CYP enzyme superfamily, but was also hypothesized to have arisen by lateral gene transfer from plants [18]. Eukaryotic CYP51s are integral membrane proteins tethered by a hydrophobic N-terminal ‘anchor’ domain. Mtb CYP51 is

Mtb P450s – insights from sequence analysis

The P450 ‘superfamily’ is classified into families and sub-families based on the extent of amino acid sequence identity. P450s with ≥40% identity are classified in the same family and often exhibit similar substrate selectivity. Currently, the only other Mtb P450 with sufficient amino acid sequence similarity to functionally characterized P450s (other than CYP51 and, possibly, CYP136) that enables any confident assignment of substrate preference is CYP132 (Table 1). This P450 is similar to

Redox partner systems for the Mtb P450s

Redox partner systems are required for P450 function. In ‘classical’ prokaryotic class I P450 systems (typified by the intensively studied P450 cam), NAD(P)H provides electrons required for oxygen activation by reducing a ferredoxin reductase (FDR), with a ferredoxin mediating electron transport between the FDR and the P450 [8]. The Mtb genome contains several candidate redox systems to support P450 function: there are several ferredoxins, with obvious redox partner systems being those that are

Structural and mechanistic characterization of key Mtb P450 isoforms

Atomic structures of two important Mtb P450s were determined recently 24, 40. Structural data for both CYP51 and CYP121 have had fundamental impacts on the understanding of P450 structure and function. The Mtb CYP51 structure was the first structure determined for this important P450 class, and structures were solved initially in complex with 4-phenylimidazole and the antifungal fluconazole [40]. These structures revealed a substantial distortion of the I helix, which is the longest helical

Inter-relationships between the Mtb P450s…or a lack of them!

With the limited availability of information regarding Mtb P450 substrate specificity and physiological function, a bioinformatics approach seems to be a logical route to derive some of these important data. The P450 superfamily is vast and the availability of numerous P450 sequences has facilitated the construction of detailed evolutionary trees that demonstrate hierarchical relationships [43]. For new P450s, this type of comparative analysis is extremely useful in recognizing related P450s

Concluding remarks and future perspectives

A clear priority for future studies of the Mtb CYPome is the determination of the roles of P450s in metabolism, cellular defence or infectivity. The genome of Mycobacterium leprae, the other major human mycobacterial pathogen and the causative agent of leprosy, retains only one functional P450 (plus several pseudogenes) and this might be taken as evidence against major roles for P450s in physiology and virulence of Mtb. The M. leprae P450 (CYP164A1) shows the highest similarity to Mtb CYP140

Acknowledgements

We thank the Medical Research Council (for a studentship to D.C.), the Biotechnology and Biological Sciences Research Council and GlaxoSmithKline (for a CASE studentship to D.G.L.), the European Commission (for the NM4TB network and a postdoctoral fellowship to K.J.M. through the X-TB network) and the Public Service Department of Malaysia and Universiti Malaysia Sarawak (UNIMAS) for a PhD studentship award (M.S.). A.W.M., D.L. and K.J.M. thank the Royal Society for the award of a Leverhulme

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