Journal of Inorganic Biochemistry

Journal of Biological Chemistry, 2010

The Mycobacterium tuberculosis cytochrome P450 enzyme CYP142 is encoded in a large gene cluster involved in metabolism of host cholesterol. CYP142 was expressed and purified as a soluble, low spin P450 hemoprotein. CYP142 binds tightly to cholesterol and its oxidized derivative cholest-4-en-3-one, with extensive shift of the heme iron to the high spin state. High affinity for azole antibiotics was demonstrated, highlighting their therapeutic potential. CYP142 catalyzes either 27hydroxylation of cholesterol/cholest-4-en-3-one or generates 5-cholestenoic acid/cholest-4-en-3-one-27-oic acid from these substrates by successive sterol oxidations, with the catalytic outcome dependent on the redox partner system used. The CYP142 crystal structure was solved to 1.6 Å , revealing a similar active site organization to the cholesterol-metabolizing M. tuberculosis CYP125, but having a near-identical organization of distal pocket residues to the branched fatty acid oxidizing M. tuberculosis CYP124. The cholesterol oxidizing activity of CYP142 provides an explanation for previous findings that ⌬CYP125 strains of Mycobacterium bovis and M. bovis BCG cannot grow on cholesterol, because these strains have a defective CYP142 gene. CYP142 is revealed as a cholesterol 27-oxidase with likely roles in host response modulation and cholesterol metabolism.

Journal of Biological Chemistry, 2009

The Journal of biological chemistry, 2017

The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands. A survey of azole antifungal drugs showed that CYP126A1 is inhibited strongly by azoles containing an imidazole ring but not by those tested containing a triazole ring. To further explore the molecular preferences of CYP126A1 and search for probes of enzyme function, we conducted a high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-li...

Angewandte Chemie International Edition, 2012

Journal of Biological Chemistry, 2013

Archives of Biochemistry and Biophysics, 2007

The human pathogen Mycobacterium tuberculosis has made a dramatic resurgence in recent years. Drug resistant and multidrug resistant strains are prevalent, and novel antibiotic strategies are desperately needed to counter Mtb's global spread. The M. tuberculosis genome sequence revealed an unexpectedly high number of cytochrome P450 (P450) enzymes , and parallel studies indicated that P450inhibiting azole drugs had potent anti-mycobacterial activity. This article reviews current knowledge of structure/function of P450s and redox partner systems in M. tuberculosis. Recent research has highlighted potential drug target Mtb P450s and provided evidence for roles of selected P450 isoforms in host lipid and sterol/steroid transformations. Structural analysis of key Mtb P450s has provided fundamental information on the nature of the heme binding site, P450 interactions with azole drugs, the biochemical nature of cytochrome P420, and novel mutational adaptations by which azole binding to P450s may be diminished to facilitate azole resistance.

Journal of Biological Chemistry, 2009

We report characterization and the crystal structure of the Mycobacterium tuberculosis cytochrome P450 CYP125, a P450 implicated in metabolism of host cholesterol and essential for establishing infection in mice. CYP125 is purified in a high spin form and undergoes both type I and II spectral shifts with various azole drugs. The 1.4-Å structure of ligand-free CYP125 reveals a "letterbox" active site cavity of dimensions appropriate for entry of a polycyclic sterol. A mixture of hexa-coordinate and penta-coordinate states could be discerned, with water binding as the 6th heme-ligand linked to conformation of the I-helix Val 267 residue. Structures in complex with androstenedione and the antitubercular drug econazole reveal that binding of hydrophobic ligands occurs within the active site cavity. Due to the funnel shape of the active site near the heme, neither approaches the heme iron. A model of the cholesterol CYP125 complex shows that the alkyl side chain extends toward the heme iron, predicting hydroxylation of cholesterol C27. The alkyl chain is in close contact to Val 267 , suggesting a substrate binding-induced lowto high-spin transition coupled to reorientation of the latter residue. Reconstitution of CYP125 activity with a redox partner system revealed exclusively cholesterol 27-hydroxylation, consistent with structure and modeling. This activity may enable catabolism of host cholesterol or generation of immunomodulatory compounds that enable persistence in the host. This study reveals structural and catalytic properties of a potential M. tuberculosis drug target enzyme, and the likely mode by which the host-derived substrate is bound and hydroxylated.

International Journal of Molecular Sciences

Cytochrome P450 monooxygenases (CYPs/P450s) are well known for their role in organisms’ primary and secondary metabolism. Among 20 P450s of the tuberculosis-causing Mycobacterium tuberculosis H37Rv, CYP128A1 is particularly important owing to its involvement in synthesizing electron transport molecules such as menaquinone-9 (MK9). This study employs different in silico approaches to understand CYP128 P450 family’s distribution and structural aspects. Genome data-mining of 4250 mycobacterial species has revealed the presence of 2674 CYP128 P450s in 2646 mycobacterial species belonging to six different categories. Contrast features were observed in the CYP128 gene distribution, subfamily patterns, and characteristics of the secondary metabolite biosynthetic gene cluster (BGCs) between M. tuberculosis complex (MTBC) and other mycobacterial category species. In all MTBC species (except one) CYP128 P450s belong to subfamily A, whereas subfamily B is predominant in another four mycobacter...

Biotechnology and Applied Biochemistry, 2020

GTP cyclohydrolase I enzyme (GTPCH-I) is a rate limiting enzyme in the biosynthesis pathway of tetrahydrobiopterin (BH4) and tetrahydrofolate (THF) compounds; latter being are an essential compounds involved in many biological functions. This enzyme has been evaluated structurally and functionally in many organisms to understand its putative role in cell processes, kinetics, regulations, drug targeting in infectious diseases, pain sensitivity in humans, and so on. In Mycobacterium tuberculosis (a human pathogen causing tuberculosis), this GTPCH-I activity has been predicted to be present in Rv3609c gene (folE) of H 37 Rv strain, which till date has not been studied in detail. In order to understand in depth, the structure and function of folE protein in M. tuberculosis H 37 Rv, in silico study was designed by using many different bioinformatics tools. Comparative and structural analysis predicts that Rv3609c gene is similar to folE protein ortholog of Listeria monocytogenes (cause food born disease), and uses zinc ion as a cofactor for its catalysis. Result shows that mutation of folE protein at 52th residue from tyrosine to glycine or variation in pH and temperature can lead to high destability in protein structure. Studies here have also predicted about the functional regions and interacting partners involved with folE protein. This study has provided clues to carry out experimentally the analysis of folE protein in mycobacteria and if found suitable will be used for drug targeting.

2011

My thanks go out to my friends and colleagues in the Munro, Scrutton, and Leys groups for all their help, discussions, and communication. Finally, I would like to thank my family, my parents, and my wife who give me a great support and understanding for my studies in Manchester.