KshAB (3-Ketosteroid 9␣-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O 2 . Nevertheless, the reactivity of KshAB with O 2 was low in the presence of 1,4-androstadiene-3,17-dione, with a k cat / K mO 2 of 2450 ؎ 80 M ؊1 s ؊1. The crystallographic structure of KshA, determined to 2.3 Å , revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroidtransforming enzyme of biotechnological and medical importance.Mycobacterium tuberculosis, arguably the world's most successful pathogen, infects one-third of the human population and has again become a global threat due in part to the emergence of extensively drug-resistant strains (XDR-TB) that are virtually untreatable with current medicines (1). Despite this alarming development, a surprising amount of the pathogen's physiology remains unknown. One recently discovered aspect of the physiology of M. tuberculosis is its cholesterol catabolic pathway (2). Studies of mutants in cholesterol uptake (3) and degradation (4) in various animal models have indicated that cholesterol catabolism is most important during the chronic phase of infection, although the latter study also provided evidence that it occurs from an early stage and contributes to dissemination of the pathogen in the host. Further study of cholesterol catabolism and the pathway enzymes are required to elucidate the precise role of cholesterol catabolism in infection.The cholesterol catabolic pathway of M. tuberculosis involves degradation of the branched alkyl side chain and the four-ringed steroid nucleus, as occurs in Rhodococcus jostii RHA1, a nonpathogenic, mycolic acid-producing actinomycete (2), although it is unclear whether the order of this degradation is obligatory. Side-chain degradation proceeds via a ␤-oxidative type process. Degradation of the steroid nucleus is initiated by 3␤-hydroxysteroi...

Mycobacterium tuberculosis (Mtb)4 infects one-third of the human population and is the leading cause of lethal bacterial infections worldwide. Synergy of Mtb with the HIV virus and the emergence of extensively drug-resistant Mtb strains (XDR-TB) have further emphasized this pathogen as a major global health threat (1). However, understanding of many of its key physiological processes, particularly those contributing to intracellular survival, is limited. One element of Mtb physiology that may prove important for therapeutic development is its unusually high number of cytochromes P450 (P450s). P450s are heme-dependent mono-oxygenases that utilize reducing equivalents from NAD(P)H relayed via electron transfer proteins to activate heme-bound O 2 . Typical of actinomycete genomes, that of Mtb includes 20 genes encoding P450s (2). In these bacteria, P450s are typically involved in the initial catabolism of growth substrates and in secondary metabolite biosynthesis. Given the high number of P450s in Mtb and their susceptibility to azole drugs, this class of enzyme has been proposed as a promising target for antimycobacterial therapeutic development (2). However, the function of most mycobacterial P450s has yet to be determined.The gene encoding one Mtb P450, cyp125 (Rv3545c), belongs to a large cluster of genes encoding cholesterol degradation (3). This cluster is conserved in several actinobacteria including the non-pathogenic soil bacterium, Rhodococcus jostii RHA1. The genes encode functions necessary for cholesterol import (4), as well as for the degradation of the steroid side chain and rings A and B (3). Several of the ring degradation enzymes from Mtb have been characterized biochemically (5-9). Animal infection studies of mutants deficient in cholesterol uptake and catabolism have indicated that cholesterol metabolism in Mtb plays an important role in infection, contributing to both dissemination in the host (7) and persistence (9, 10). These results are consistent with several mutant screens, which previously identified numerous genes in the cholesterol degradation pathway as having an impact on intracellular growth and survival in both mouse and macrophage models (11, 12), implicating cholesterol catabolism in Mtb pathogenicity.Cyp125 has been implicated in the in vivo survival of Mtb, although its catalytic activity has not been demonstrated, and its physiological role remains unclear. The cyp125 gene is upregulated during growth of Mtb in interferon ␥-activated macrophages (13)

SummaryThe cyp125 gene of Rhodococcus jostii RHA1 was previously found to be highly upregulated during growth on cholesterol and the orthologue in Mycobacterium tuberculosis (rv3545c) has been implicated in pathogenesis. Here we show that cyp125 is essential for R. jostii RHA1 to grow on 3-hydroxysterols such as cholesterol, but not on 3-oxo sterol derivatives, and that CYP125 performs an obligate first step in cholesterol degradation. The involvement of cyp125 in sterol side-chain degradation was confirmed by disrupting the homologous gene in Rhodococcus rhodochrous RG32, a strain that selectively degrades the cholesterol side-chain. The RG32Wcyp125 mutant failed to transform the side-chain of cholesterol, but degraded that of 5-cholestene-26-oic acid-3b-ol, a cholesterol catabolite. Spectral analysis revealed that while purified ferric CYP125 RHA1 was < 10% in the low-spin state, cholesterol (KD app = 0.20 Ϯ 0.08 mM), 5a-cholestanol (KD app = 0.15 Ϯ 0.03 mM) and 4-cholestene-3-one (KD app = 0.20 Ϯ 0.03 mM) further reduced the low spin character of the haem iron consistent with substrate binding. Our data indicate that CYP125 is involved in steroid C26-carboxylic acid formation, catalysing the oxidation of C26 either to the corresponding carboxylic acid or to an intermediate state.

Background: Mycobacterium tuberculosis (Mtb) degrades cholesterol throughout its infection cycle. Results: Cholesterol ring-degrading enzymes have higher activities with side chain degradation intermediates than with compounds with fully degraded side chains. Conclusion: Cholesterol side chain and ring degradation occur concurrently. Significance: Understanding bacterial cholesterol catabolism facilitates the design of novel therapeutics and the production of high value steroids.