The preponderance of P450s in the Mycobacterium tuberculosis genome

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The preponderance of P450s in the Mycobacterium tuberculosis genome Kirsty J. McLean, Daniel Clift, D. Geraint Lewis, Muna Sabri, Philip R. Balding, Michael J. Sutcliffe, David Leys and Andrew W. Munro Manchester Interdisciplinary Biocentre, School of Chemical Engineering and Analytical Science and School of Life Sciences, University of Manchester, Jackson’s Mill, Sackville Street, Manchester, UK, M60 1QD

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.

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 health. The resurgence of Mtb as the leading cause of human mortality among infectious diseases is, in part, a result of synergy with the HIV virus (whereby Mtb thrives in immune-compromised HIV-infected individuals) and partly because of the development and proliferation of Mtb strains that are resistant to existing antitubercular drugs [2,3]. The severity of the situation was recognized by the World Health Organization, which described the spread of multidrug-resistant Mtb strains as a ‘global emergency’. The Mtb genome sequence revealed several unusual features. Novel protein families were identified, notably the PE and PPE families. These are glycine-rich proteins encoded by clustered genes that comprise up to 10% Corresponding author: Munro, A.W. ([email protected]). Available online 3 April 2006

of the Mtb genome [4,5]. A large proportion of the genome (w250 enzymes, versus only w50 in the similarly sized Escherichia coli genome) is devoted to the production of proteins involved in lipid synthesis and metabolism [1]. This partly reflects the enormous diversity of lipophilic molecules produced by Mtb, including the highly complex mycolic acids that form key cell envelope components [6]. The Mtb cell wall is an intricate structure that provides a formidable barrier to entry of molecules into the cell and is a key determinant of both Mtb pathogenicity and resistance to attack by the host [7]. An unexpected finding was that 20 cytochrome P450 enzymes (P450s) were encoded by Mtb. Previous prokaryotic genome sequences had revealed fewer P450s and E. coli has none (but can produce heterologously expressed P450s at high levels) [8]. Early characterization of prokaryotic P450s focused on systems that were often associated with catabolic pathways, such as the camphor hydroxylase P450 cam (CYP101) from Pseudomonas putida [9]. This gave the misleading impression that, although P450s are essential for eukaryotic physiology, prokaryotic P450s might be dispensable ‘luxury items’ that are necessary only when regular nutrient sources are scarce. The sequence of the Mtb genome strongly indicated that this was not the case and subsequent genome sequences of related actinobacteria [e.g. Mycobacterium smegmatis (39 P450s) and Streptomyces coelicolor (18 P450s)] showed that a large P450 component was a feature of several prokaryotic genomes. In Streptomycetes, P450s are important for secondary metabolite production, including antibiotics. These Gram-positive bacteria have a high CYP gene density relative to other organisms. For example, the human genome (w3000 Mb) encodes 57 P450s, whereas Mtb has 20 P450s in just 4.41 Mb [10]. Thus, the relative ‘CYP density’ is w240-fold greater in the Mtb genome, which suggests crucial roles for many of these isoforms and indicates that Mtb might rely heavily on P450 monooxygenase chemistry. Among the Mtb P450s was a representative of the sterol demethylase class (CYP51 family), a P450 type that was previously thought to be exclusively eukaryotic [11]. Importantly, this enzyme is a key antifungal drug target. Fungal CYP51s are inhibited by drugs such as fluconazole, ketoconazole and voriconazole [12]. These agents have high affinity for the hydrophobic CYP51 active site and inactivate haem

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redox function by coordinating the haem iron through an imidazole or triazole nitrogen atom. This binding mode hinders substrate access to the haem and prevents interaction of the haem iron with oxygen. Because of the success of azoles as antifungals in clinical use, the possibility of targeting Mtb P450s with novel drugs is an exciting possibility. This whetted the appetite for researchers in the P450 and Mtb fields to examine in more detail the P450 systems in this bacterium. This review discusses the current level of understanding of structural and functional properties of the Mtb P450 complement, and considers the evolution and potential of Mtb P450s as drug targets. 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 cycle (i.e. the catalytically relevant oxidants that attack the substrate) are transient; therefore, compelling structural and spectroscopic evidence for their formation remains somewhat elusive. The roles of human adrenal P450s in steroid synthesis and of multiple hepatic isoforms of P450s in xenobiotic metabolism have been intensively studied and the importance of P450-dependent metabolism in human ROH

RH Fe3+ (i)

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Fe3+ RH

1st e–

Fe3+ ROH

(ii)

(vii) (vi)

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Fe2+ RH

H+

H2 O

(iii)

O2

(Fe3+–OOH) RH (v) H+

(iv) Fe3+– O22– RH

Fe3+– O2– RH 2nd e–

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Figure 1. The catalytic cycle of cytochrome P450. The nature of intermediate species in the catalytic cycle of P450s has been the subject of intensive study worldwide since the discovery of P450 in the 1950s. (i) The resting ferric (Fe3C) form binds to substrate (RH) and (ii) is reduced by a single electron (eK) to the ferrous (Fe2C) state before (iii) binding of molecular oxygen (O2). (iv) Delivery of a second electron from the redox partner and two protons (likely to be from bulk solvent in most cases, although transfer is probably mediated by active site amino acid side chains) results in the formation of transient, reactive species that catalyse oxygenation of substrate (v–vii). The product (ROH) dissociates to return the haem to its resting state (viii). www.sciencedirect.com

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physiology is well characterized [15]. In bacteria, the P450s are used in catabolic pathways for the breakdown of recalcitrant pollutants and for the synthesis of polyketide antibiotics and other complex macromolecules, for example [16]. The ability of P450s to catalyse regio- and stereo-selective oxidation of organic molecules has captured the interest of the biotechnology industry. The exploitation of P450 chemistry provides cleaner and more efficient routes to oxygenation of organic compounds, for example, for the production of steroids, bioactive lipids and chiral synthons [17]. 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 devoid of a membrane anchor, which is typical for prokaryotic P450s. CYP51 has been expressed in E. coli, purified and characterized biochemically and structurally (see later). Preliminary studies indicated that Mtb CYP51 could demethylate sterols (lanosterol, dihydrolanosterol and the plant sterol obtusifoliol) when coupled to heterologous redox partners [11]. An early study indicated that cholesterol was present in mycobacteria and that a sterol biosynthetic pathway might exist in Mtb. However, cholesterol was later discovered to be a component of the growth medium used in the study [19]. The absence of key sterol-synthesis pathway enzymes in the genome of Mtb also indicated that it is incapable of de novo sterol synthesis. However, cholesterol is required for Mtb infectivity in humans and, thus, the sterol binding activity of CYP51 could be an important factor that enables the pathogen to establish itself in the host [20]. Whereas most of the Mtb P450s show limited amino acid sequence similarity to each other (see later), another Mtb P450 (CYP136) has significant similarity to Mtb CYP51 in addition to eukaryotic CYP51s and various fatty acidbinding P450s. However, whether or not CYP136 also has a role in the binding and/or metabolism of host sterols or steroids will not be elucidated until it is functionally characterized. Mtb P450s – insights from sequence analysis The P450 ‘superfamily’ is classified into families and subfamilies based on the extent of amino acid sequence identity. P450s with R40% 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 bacterial fatty acid oxygenases and to eukaryotic CYP4 family P450s (fatty acid u-hydroxylases) [21]. Preliminary studies on CYP132 demonstrate that fatty acids and azole

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Table 1. Key facts about P450s and redox partners from Mycobacterium tuberculosis H37Rva Mtb gene Rv0764c

Protein name CYP51

Rv2276

CYP121

Rv1394c Rv2268c Rv2266

CYP132 CYP128 CYP124

Rv3545c Rv3059 Rv1777 Rv1256c Rv1321 Rv3106

CYP125 CYP136 CYP144 CYP130 CYP141 FprA

Rv0886

FprB

Rv3554

FdxB

Rv0688 Rv0763c

FdR Fdx

Rv1786

Rv1786

Rv2007c

FdxA

Rv1177

FdxC

Key facts Sterol demethylase P450. Fluconazole-bound structure solved. Role in host steroid inactivation? Highest resolution P450 structure. Novel azole binding mode. Nanomolar azole binding and positive correlation of azole Kd and mycobacterial MIC values. Related to fatty acid metabolising P450s. Role in Mtb virulence? Required for optimal growth of Mtb in vitro. Similarities in substrate-binding regions to P450s involved in antibiotic synthesis. Induced in macrophages. Needed for survival in infected mice. Weakly related to sterol demethylase P450 family. Exhibits tight binding of azole antifungal drugs. P450 absent from Mycobacterium bovis genome. P450 absent from Mycobacterium bovis genome. Structurally resolved adrenodoxin reductase homologue. Supports Mtb P450 function. Putative FprA–ferredoxin fusion, which might function as a single component P450 redox partner. Putative ferredoxin reductase–ferredoxin fusion redox partner for Mtb P450s. Ferredoxin reductase. Supports CYP51 function. Chromosomally adjacent to CYP51. Validated ferredoxin redox partner for CYP51. Chromosomally adjacent to CYP143 and probable ferredoxin redox partner. Putative ferredoxin. Induced by various stresses, including those experienced by Mtb in host cells. Putative ferredoxin required for optimal growth in vitro.

Refs [11,20,40] [24–26]; unpublishedb

[22] [49] N/A [50–52] N/A N/A [47] [47] [11,32,33] N/A N/A [39] [11] N/A [28,29,53–55] [49]

a

Abbreviation: N/A, not available. b H.E. Seward et al., unpublished.

derivatives interact with CYP132, but its physiological role remains unclear. However, the gene that encodes CYP132 (Rv1394c) is adjacent to a regulator of the AraC class (Rv1395), and a knockout of this regulator attenuated Mtb in a mouse model. The regulator repressed its own transcription but induced transcription of the P450, which implicates CYP132 in bacterial virulence [22]. Further insights into the relative importance of different Mtb P450s and their redox partners come from genomic analyses (Box 1). Several Mtb P450s exhibit similarity to bacterial P450s that participate in the synthesis of polyketide and other antibiotics (e.g. P450 eryF from Saccharopolyspora erythraea and P450 NanP from Streptomyces nanchangensis, which are involved in the manufacture of erythromycin and the polyether ionophore nanchangmycin, respectively) [16,23]. However, low levels of overall identity preclude accurate diagnosis of function based simply on sequence comparisons. One such Mtb P450 is the structurally characterized CYP121 [24]. Further evidence pointing to the ability of CYP121 to bind complex polycyclic molecules comes from its high affinity for several bulky azole antifungals. Indeed, CYP121 binds to these azoles tighter than CYP51 does – despite the fact that CYP51 is a member of the P450 target family for these drugs [25]. The same azoles inhibit M. smegmatis cell growth (with MIC values at least as low as those for leading anti-Mtb drugs), and are also active against Mtb [26]. Given that there are 20 Mtb P450s, a number of which are already characterized to have high affinity for these inhibitors (including CYP121, CYP132, CYP144 and CYP51), there is a strong possibility that azoles can be www.sciencedirect.com

Box 1. Genomic analysis of Mtb P450 systems In the search for information about the various Mtb P450s and associated redox systems, important clues have been revealed by gene array and genomic insertional mutagenesis strategies. These suggest involvement of selected P450 systems in processes such as cell viability and infectivity. Genome-wide transposon mutagenesis revealed several genes that are required for optimal mycobacterial growth under laboratory conditions, including both the genes that encode FdxC and CYP128 [49]. Hypoxia (which is associated with Mtb persistence in the latent state) and acidity (which mimics the environment of phagocytosed cells) induce FdxA expression [28,29], which is controlled by the transcriptional regulator DevR (or DosR). The dosR regulon contains several genes that respond to signals including oxygen levels and nitric oxide [53]. However, because acidity does not upregulate dosR, a different mechanism must operate to induce FdxA at low pH. Thus, FdxA seems to be a key protein in general Mtb stress responses. FdxA is also induced by heat shock and by treatment with thiolactomycin and isoniazid – drugs that inhibit mycolic acid synthesis in Mtb – and by other antibacterials [50,54,55]. In one study, the gene encoding CYP121 and adjacent genes were deleted in two of 100 Mtb clinical isolates studied by sequencing and microarray analysis [56]. However, it is not clear whether the deletion was moderately deleterious or actually provided a selective advantage to the pathogen. Clearly, the complexity of data produced by numerous microarray studies can be overwhelming and can conflict with other results in the literature [51]. A consensus about which genes are most important to Mtb can be reached from the recognition of genes as important and/or critical in two or more diverse studies of differential expression or gene essentiality. By this criterion, CYP125 (Rv3545c) and its operon are highlighted because they are induced during Mtb engulfment in macrophages and are required for Mtb survival in infected mice [51,52,57]. Despite some uncertainties about the absolute diagnostic capacity of such studies, they certainly provide important clues to proteins and regulatory systems that have pivotal roles in bacterial viability and pathogenesis.

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used in anti-tuberculosis therapy, possibly to exploit a ‘multi-hit’ strategy whereby single azole-based drugs inactivate the function of multiple Mtb P450 isoforms (Table 1). 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 chromosomally adjacent to P450s. Two such examples exist in Mtb; first, the 3Fe–4S ferredoxin Fdx (adjacent to CYP51) was shown to support the sterol demethylase activity of CYP51 [11]. Second, the 3Fe–4S ferredoxin encoded by Rv1786 is adjacent to CYP143. The genes encoding the latter pair of proteins are located in a region that is close to several PE and PPE genes, and to esat-6 family genes. Esat-6 (the product of gene esxA) is an infectivity-related protein that is deleted from the genome of the tuberculosis vaccine strain Mycobacterium bovis bacille Calmette-Gue´rin (BCG) [27]. Other potential redox partners include FdxA (Rv2007c), a ferredoxin that might bind both 4Fe–4S and 3Fe–4S cofactors. FdxA transcription is induced by hypoxia and upregulated by low pH in vitro [28,29], so acidic conditions might mimic the environment that is encountered by Mtb cells phagocytosed by host immune cells (Box 1). Rv1177 could be a gene that is important for optimal Mtb growth: it encodes the ferredoxin FdxC, which has a similar cofactor content to FdxA. Rv3503c also encodes a putative 3Fe–4S ferredoxin (FdxD). In the search for potential NAD(P)H-dependent flavincontaining reductases, one candidate stands out immediately. In eukaryotic mitochondrial P450 systems, a prokaryotic-like class I redox system operates, which involves the FAD-containing adrenodoxin reductase (ADR) and the 2Fe–2S adrenodoxin [30]. The use of such a system is consistent with the endosymbiont theory of mitochondrial evolution, particularly because non-mitochondrial eukaryotic P450s usually source electrons from a single FAD- and FMN-containing enzyme (NADPH– cytochrome P450 reductase) [31]. In Mtb, flavoprotein reductase A (FprA) is an ADR homologue, which is evident from both its amino acid sequence and from its atomic structure [32]. It is reduced by both NADPH and NADH, although NADPH binding is tighter. NADPH reduces the FprA FAD cofactor to its semiquinone (single electron reduced) form, whereas NADH reduces it fully to hydroquinone [33]. This unusual phenomenon might be associated with differential binding modes and/or affinities of reduced forms of FprA for NAD(P)H. FprA is a soluble enzyme that transfers electrons to Mtb Fdx and, thus, is a viable Mtb P450 partner enzyme. Elsewhere in the genome, FprB is a FprA–Fdx-like fusion protein. The protein remains uncharacterized, but it is tempting to speculate that this single protein functions as a surrogate www.sciencedirect.com

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for the two-component ferredoxin reductase–ferredoxin system to provide a more efficient system for electron transfer to Mtb P450s. Similar ‘streamlining’ strategies, which involve fusing P450 redox partners and usually result in enhanced catalytic activity, have been adopted by other bacteria [34]. FdxB is another putative ferredoxin reductase–ferredoxin fusion in which the ferredoxin is likely to bind to a 2Fe–2S cluster. Ferredoxins with 2Fe– 2S, 3Fe–4S and 4Fe–4S clusters have all been shown to interact productively with P450s in other bacteria [35–37]. Other potential FAD-containing ferredoxin reductase redox partners are encoded by Rv0688 and Rv1869c genes. In the case of Rv0688, some similarities with the primary structure of Rhodococcus phthalate dioxygenase reductase (PDR) strike resonance given the recent characterization of natural P450–PDR fusion proteins (with potential detoxification roles) in this genus and in other bacteria [38]. The FAD-containing reductase protein product of Rv0688 (termed FdR) also couples productively with Fdx and CYP51 to reconstitute lanosterol demethylation [39] (Box 1). 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 segment in CYP51. In all structurally characterized P450s, the I helix is rich in residues that are involved in substrate binding and catalysis, but its degree of displacement in CYP51 is the largest observed in structures of P450s (Figure 2). The N-terminal portion of the helix twists away from the structural core of the protein, making an angle of 1458 between the N- and C-terminal segments. This distortion releases another conserved P450 structural element, the BC loop, from a closed conformation. This exposes a potential substrate entry and/or exit cavity that involves both elements, which runs parallel with the haem plane and extends from the active site to the protein surface. In the two most intensively studied bacterial P450s (P450 cam and the Bacillus megaterium fatty acid hydroxylase P450 BM3), the defined substrate-access channels (involving F and G helices and their interconnecting loop) run approximately perpendicular to the haem plane. Examination of the CYP51 structures indicates that both of these types of access channels should be accessible, but that conformational changes required for opening of one would necessitate closure of the other. An attractive model is that synchronization of these motions during catalysis enables substrate entry through one channel and product exit through the other [40]. Subsequent structures of ligand-free and estriol-bound Mtb CYP51 provided further insights into structural dynamics.

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(a)

(b)

(c)

(d)

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Figure 2. Atomic structures of Mtb P450s CYP51 and CYP121. (a) Overall structure and topology of the fluconazole-bound form of Mtb CYP51. The large deviation in the I helix (green) is evident. Fluconazole (yellow, blue and magenta) is depicted in spacefill above the haem (red) for CYP51. (b) A similar representation of the structure of ligand-free CYP121, with a straight I helix. Parts (c) and (d) show close-ups of the immediate haem environments in the P450s. (c) Direct coordination of the CYP51 haem iron (N-Fe) by fluconazole; and (d) the interactions of Arg386 and the I helix residue Ser237 with the water ligand to the haem iron in CYP121, which leads to stabilization of the low-spin haem iron form of CYP121 [24,40].

In the ligand-free form, the C helix and portions of the adjacent BC loop (which define a substrate entry site) are disordered, which leads to further opening of the substrate access site. However, binding of estriol and azoles produces conformational change and ordering of the C helix. This was interpreted as a ‘lid closing’ event, which completes the formation of a high-affinity CYP51 substrate-binding site [41]. Several residues associated with resistance to and/or reduced affinity for azoles have been recognized in clinical isolates of pathogens (e.g. Candida albicans). Importantly, the corresponding residues in Mtb CYP51 are largely located in dynamic regions and/or regions that affect inter-domain conformational changes, as opposed to those that affect substrate and/or drug binding per se. It can be concluded that mutations causing diminished drug interactions are disfavoured because of adverse affects on substrate binding and, thus, that most clinical mutations affect protein dynamics and conformational events instead, which leads to weakened azole affinity [41]. Mtb CYP121 provided the highest resolution P450 ˚ and gave further insights into P450 structure at 1.06 A architecture and ligand binding. Structures of ligand-free and iodopyrazole-bound forms revealed that the haem itself was bound in two distinct orientations, related by a 1808 rotation about the CHa-Fe-CHd axis [24]. This is probably a common phenomenon in P450s but has previously gone unrecognized because of the lower resolution of preceding structures. The positioning of the www.sciencedirect.com

ligand iodopyrazole (stacking with aromatic residues in a channel between F and G helices) also defined a potential substrate entry–exit port in a similar region to that seen for Mtb CYP51. However, the most surprising phenomenon was associated with the organization of amino acids that define the active site cavity. The active site is spatially restricted: dominant features in the immediate haem vicinity include a strong hydrogen-bonding network that involves Ser237, Arg386 and a water molecule bound to the distal (sixth coordination position) of the iron (Figure 2). This arrangement restricts access to large molecules and suggests that dramatic conformational changes (involving breakage and reorganization of the network) are required to facilitate substrate binding in a catalytically relevant mode. This also raises important questions regarding binding modes for various bulky azoles, many of which have high affinity for CYP121 [25]. Recently, the structure of a CYP121-fluconazole complex has been resolved. Preliminary data indicate that azoles are accommodated by a novel binding mode (H.E. Seward et al., unpublished). Thus, the fluconazolebound structures of both CYP51 and CYP121 could provide important lessons in understanding the nature of azole binding, and how azole resistance develops. This, in turn, might facilitate a more ‘intelligent’ design strategy for next-generation P450 inhibitors. Several older azoles cannot be used systemically because of adverse reactions with human P450s and other proteins. For example, a study of transcriptional responses of Mtb to metabolic inhibitors indicated that clotrimazole and

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econazole had no effect on oxygen-dependent respiration but suggested non-specific interactions with succinate dehydrogenase [42]. However, newer-generation azoles (e.g. fluconazole and voriconazole) are systemically tolerated and can clearly provide the ‘templates’ for the design of antitubercular derivatives. 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 and to ascertain the substrate class that is favoured. For the Mtb P450 complement, protein alignments and evolutionary analysis reveal a surprising lack of similarity. Figure 3 shows a ‘neighbour-joining’ evolutionary tree for Mtb P450s, including selected ‘reference’ P450s from other organisms. These reference P450s either exhibit higher levels of similarity to individual Mtb P450s than do the other Mtb isoforms, or demonstrate the short branch lengths expected for highly related P450s. This analysis demonstrates the extent of divergence between the Mtb P450s, particularly when compared with the short branch lengths between the mammalian CYP2C family members (w72–78% identical) and CYP4 fatty acid hydroxylases (e.g. 90% identity between rabbit CYP4A5 and CYP4A6). Among the Mtb P450s, CYP132 has the strongest similarity to the wellcharacterized eukaryotic fatty acid oxygenating P450s. However, it is evolutionarily distant from them and has a lower level of identity to them than does the Bacillus megaterium P450 BM3 fatty acid hydroxylase [14,34]. Another example of the extensive divergence of Mtb P450s comes from comparison of the degree of similarity between Mtb CYP51 and its closest Mtb relative, CYP136, with that for Mtb CYP51 and eukaryotic sterol demethylases. Even the only two Mtb P450s formally classified in the same family (CYP135A1 and CYP135B1) have limited similarity (40% identity). What is the significance of this extraordinary dissimilarity among Mtb P450s? Potentially, it reflects that there is little functional redundancy and that the 20 isoforms originated by duplications and divergent evolution from a progenitor millions of years ago. However, a progenitor is not obvious in the Mtb P450 cluster. An alternative might be that Mtb has ‘hijacked’ several P450 genes and assimilated them into its genome, thereafter evolving them to suit cellular requirements. A previous study indicates that Mtb contains several genes of eukaryotic origin [44]. Many or all of these genes could have been incorporated by horizontal gene transfer from eukaryotic hosts. P450s such as CYP51 could have arrived in Mtb by such a route, whereas others could have been transferred from prokaryotes. One potential benefit from this strategy (possibly involving at least CYP51) could be that Mtb has www.sciencedirect.com

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furnished its genome with the metabolic capacity to inactivate host sterols and/or steroids to prolong the infective state [44]. 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 but might not be a direct homologue [45]. However, M. leprae has undergone substantial gene decay during its evolution and is effectively an obligate parasite of its host [46]. Strong conservation of a large P450 pool in other mycobacteria (and in related genera) suggests that clusters of these mono-oxygenases have pivotal roles in the biology of this genus. The M. bovis genome sequence revealed extremely close evolutionary relationships with Mtb [47]. However, the two Mtb P450-encoding genes Rv1256c (CYP130) and Rv1321 (CYP141) are absent from the RD12 and RD13 segments of the M. bovis BCG strain used in the tuberculosis vaccine (RD represents ‘region of difference’ between the Mtb and M. bovis genomes). These P450s are also absent from the genome of the native M. bovis strain [46]. However, whether this defines these genes as non-essential for normal mycobacterial metabolism or crucial to the peculiar biochemistry of Mtb has yet to be resolved. On the basis of many preceding studies on diverse P450s, it can safely be concluded that Mtb P450 substrates are mainly hydrophobic in nature. This seems to be consistent with the fact that Mtb emphasises lipid metabolism and with the occurrence of complex lipids in the Mtb cell envelope [7]. Because of the strong interactions that have been demonstrated between azole drugs and various Mtb P450s, it might be considered that administration of azoles to cultured Mtb (at sub-MIC levels) would lead to defects in Mtb lipid metabolism. This, in turn, might enable links to be made between P450 function and specific lipid products. This strategy was applied successfully recently for M. smegmatis. The azole drugs clotrimazole and econazole inhibit synthesis of all types of glycopeptolipid, possibly by inhibiting one or more P450s involved in the hydroxylation of amide-linked fatty acid precursor(s) of glycopeptolipids [48]. Structural data for Mtb P450s (CYP51 and CYP121) have provided important and, perhaps, unexpected insights into P450 structure and mechanism. With respect to understanding the modes of action of azole drugs, which are the best-characterized and most clinically effective of P450 inhibitors, these structures also provide important lessons for understanding modes of P450 binding to these drugs and how resistance to these antibiotics can arise. In the case of antibiotic resistance, the possibility that mutations that affect

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Mon D NanP CYP124 CYP125A2 CYP125 CYP126 CYP142 CYP123 CYP130 CYP143 P450 cam CYP128 CYP144 CYP121 CYP141 P450 eryF CYP140 CYP154C1 CYP154C2 CYP154A1 CYP154A2 CYP154B1 CYP154B2 CYP2C9 CYP2C8 CYP2C5 Obtusifoliol14-α demethylase CYP51 Eburicol14-α demethylase Lanosterol14-α demethylase CYP136 CYP4A6 (rabbit) CYP4A5 (rabbit) CYP4F (human) P450 BM3 CYP132 CYP139 CYP138 CYP137 CYP135A1 CYP135B1 TRENDS in Microbiology

Figure 3. Evolutionary analysis of Mtb P450 enzymes. The Mtb P450s show limited evolutionary relationships as demonstrated by the evolutionary tree shown, which combines the Mtb P450s (red text) with other selected members of the P450 superfamily. Mtb CYP51 clusters with the eukaryotic sterol 14-a demethylase group but stands alone as the only Mtb P450 for which a functional assignment can clearly be made on the basis of these data (purple group). CYP136 is distantly related but potentially of similar evolutionary origin. CYP132 is also distantly related to eukaryotic and prokaryotic fatty acid hydroxylases (green group). CYP135A1 and CYP135B1 are the only two Mtb P450s classified in the same family and share precisely 40% identity (light blue group). Comparisons of only active site sequence regions that are considered important for substrate selectivity determination (substrate-recognition sequences or SRS) revealed similarities between Mtb CYP124 and the P450s MonD (from Streptomyces cinnamonensis) and NanP (from Streptomyces nanchengensis), which are involved in production of the polyether ionophore antibiotics monensin and nanchangmycin. CYP124 is shown clustered with these enzymes (dark blue group). For comparison, the close relatedness of mammalian CYP2C enzymes (gold group) and slightly more distant evolutionary relationships between members of the CYP154 family from different Streptomyces species (red group) are shown. Other structurally and functionally characterized bacterial P450s (P450 eryF and P450 cam) are included to further demonstrate the limited relatedness to these enzymes.

protein dynamics could impact on azole affinity is an important new concept that will provide formidable challenges for the rational design of novel inhibitors. However, if P450s do prove to be viable new antibiotic www.sciencedirect.com

targets in multidrug-resistant Mtb, then radical new strategies for drug development might be those that ultimately achieve victory against one of humankind’s oldest and omnipresent enemies.

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TRENDS in Microbiology

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 Trust senior research fellowship (A.W.M.) and a university research fellowship (D.L.).

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