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Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis.

ABSTRACT: The gene encoding the cytochrome P450 CYP121 is essential for Mycobacterium tuberculosis. However, the CYP121 catalytic activity remains unknown. Here, we show that the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) binds to CYP121, and is efficiently converted into a single major product in a CYP121 activity assay containing spinach ferredoxin and ferredoxin reductase. NMR spectroscopy analysis of the reaction product shows that CYP121 catalyzes the formation of an intramolecular C-C bond between 2 tyrosyl carbon atoms of cYY resulting in a novel chemical entity. The X-ray structure of cYY-bound CYP121, solved at high resolution (1.4 A), reveals one cYY molecule with full occupancy in the large active site cavity. One cYY tyrosyl approaches the heme and establishes a specific H-bonding network with Ser-237, Gln-385, Arg-386, and 3 water molecules, including the sixth iron ligand. These observations are consistent with low temperature EPR spectra of cYY-bound CYP121 showing a change in the heme environment with the persistence of the sixth heme iron ligand. As the carbon atoms involved in the final C-C coupling are located 5.4 A apart according to the CYP121-cYY complex crystal structure, we propose that C-C coupling is concomitant with substrate tyrosyl movements. This study provides insight into the catalytic activity, mechanism, and biological function of CYP121. Also, it provides clues for rational design of putative CYP121 substrate-based antimycobacterial agents.

SUBMITTER: Belin P

PROVIDER: S-EPMC2678619 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Substrate and reaction specificity of Mycobacterium tuberculosis cytochrome P450 CYP121: insights from biochemical studies and crystal structures.

Project description:Cytochrome P450 CYP121 is essential for the viability of Mycobacterium tuberculosis. Studies in vitro show that it can use the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) as a substrate. We report an investigation of the substrate and reaction specificities of CYP121 involving analysis of the interaction between CYP121 and 14 cYY analogues with various modifications of the side chains or the diketopiperazine (DKP) ring. Spectral titration experiments show that CYP121 significantly bound only cyclodipeptides with a conserved DKP ring carrying two aryl side chains in l-configuration. CYP121 did not efficiently or selectively transform any of the cYY analogues tested, indicating a high specificity for cYY. The molecular determinants of this specificity were inferred from both crystal structures of CYP121-analog complexes solved at high resolution and solution NMR spectroscopy of the analogues. Bound cYY or its analogues all displayed a similar set of contacts with CYP121 residues Asn(85), Phe(168), and Trp(182). The propensity of the cYY tyrosyl to point toward Arg(386) was dependent on the presence of the DKP ring that limits the conformational freedom of the ligand. The correct positioning of the hydroxyl of this tyrosyl was essential for conversion of cYY. Thus, the specificity of CYP121 results from both a restricted binding specificity and a fine-tuned P450 substrate relationship. These results document the catalytic mechanism of CYP121 and improve our understanding of its function in vivo. This work contributes to progress toward the design of inhibitors of this essential protein of M. tuberculosis that could be used for antituberculosis therapy.

| S-EPMC3682536 | biostudies-literature

Characterization of active site structure in CYP121. A cytochrome P450 essential for viability of Mycobacterium tuberculosis H37Rv.

Project description:Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target.

| S-EPMC2662266 | biostudies-literature

Reaction of Mycobacterium tuberculosis cytochrome P450 enzymes with nitric oxide.

Project description:During the initial growth infection stage of Mycobacterium tuberculosis (Mtb), (*)NO produced by host macrophages inhibits heme-containing terminal cytochrome oxidases, inactivates iron/sulfur proteins, and promotes entry into latency. Here we evaluate the potential of (*)NO as an inhibitor of Mtb cytochrome P450 enzymes, as represented by CYP130, CYP51, and the two previously uncharacterized enzymes CYP125 and CYP142. Using UV-visible absorption, resonance Raman, and stopped-flow spectroscopy, we investigated the reactions of (*)NO with these heme proteins in their ferric resting form. (*)NO coordinates tightly to CYP125 and CYP142 (submicromolar) and with a lower affinity (micromolar) to CYP130 and CYP51. Anaerobic reduction of the ferric-NO species with sodium dithionite led to the formation of two spectrally distinct classes of five-coordinate ferrous-NO complexes. Exposure of these species to O(2) revealed that the ferrous-NO forms of CYP125 and CYP142 are labile and convert back to the ferric state within a few minutes, whereas ferrous CYP130 and CYP51 bind (*)NO almost irreversibly. This work clearly indicates that, at physiological concentrations (approximately 1 microM), (*)NO would impair the activity of CYP130 and CYP51, whereas CYP125 and CYP142 are more resistant. Selective P450 inhibition may contribute to the inhibitory effects of (*)NO on Mtb growth.

| S-EPMC2681326 | biostudies-literature

The Mycobacterium tuberculosis cytochrome P450 system.

Project description:Tuberculosis remains a leading cause of human mortality. The emergence of strains of Mycobacterium tuberculosis, the causative agent, that are resistant to the major frontline antitubercular drugs increases the urgency for the development of new therapeutic agents. Sequencing of the M. tuberculosis genome revealed the existence of 20 cytochrome P450 enzymes, some of which are potential candidates for drug targeting. The recent burst of studies reporting microarray-based gene essentiality and transcriptome analyses under in vitro, ex vivo and in vivo conditions highlight the importance of selected P450 isoforms for M. tuberculosis viability and pathogenicity. Current knowledge of the structural and biochemical properties of the M. tuberculosis P450 enzymes and their putative redox partners is reviewed, with an emphasis on findings related to their physiological function(s) as well as their potential as drug targets.

| S-EPMC2812603 | biostudies-literature

Structural characterization of CYP144A1 - a cytochrome P450 enzyme expressed from alternative transcripts in Mycobacterium tuberculosis.

Project description:Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis (TB). The virulent Mtb H37Rv strain encodes 20 cytochrome P450 (CYP) enzymes, many of which are implicated in Mtb survival and pathogenicity in the human host. Bioinformatics analysis revealed that CYP144A1 is retained exclusively within the Mycobacterium genus, particularly in species causing human and animal disease. Transcriptomic annotation revealed two possible CYP144A1 start codons, leading to expression of (i) a "full-length" 434 amino acid version (CYP144A1-FLV) and (ii) a "truncated" 404 amino acid version (CYP144A1-TRV). Computational analysis predicted that the extended N-terminal region of CYP144A1-FLV is largely unstructured. CYP144A1 FLV and TRV forms were purified in heme-bound states. Mass spectrometry confirmed production of intact, His6-tagged forms of CYP144A1-FLV and -TRV, with EPR demonstrating cysteine thiolate coordination of heme iron in both cases. Hydrodynamic analysis indicated that both CYP144A1 forms are monomeric. CYP144A1-TRV was crystallized and the first structure of a CYP144 family P450 protein determined. CYP144A1-TRV has an open structure primed for substrate binding, with a large active site cavity. Our data provide the first evidence that Mtb produces two different forms of CYP144A1 from alternative transcripts, with CYP144A1-TRV generated from a leaderless transcript lacking a 5'-untranslated region and Shine-Dalgarno ribosome binding site.

| S-EPMC4880925 | biostudies-literature

Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1.

Project description: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-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands "moonlight" as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in M. tuberculosis, suggesting that further investigations will reveal its diverse substrate selectivity.

| S-EPMC5270475 | biostudies-literature

Structural basis for ligand promiscuity in cytochrome P450 3A4.

Project description:Cytochrome P450 (CYP) 3A4 is the most promiscuous of the human CYP enzymes and contributes to the metabolism of approximately 50% of marketed drugs. It is also the isoform most often involved in unwanted drug-drug interactions. A better understanding of the molecular mechanisms governing CYP3A4-ligand interaction therefore would be of great importance to any drug discovery effort. Here, we present crystal structures of human CYP3A4 in complex with two well characterized drugs: ketoconazole and erythromycin. In contrast to previous reports, the protein undergoes dramatic conformational changes upon ligand binding with an increase in the active site volume by >80%. The structures represent two distinct open conformations of CYP3A4 because ketoconazole and erythromycin induce different types of coordinate shifts. The binding of two molecules of ketoconazole to the CYP3A4 active site and the clear indication of multiple binding modes for erythromycin has implications for the interpretation of the atypical kinetic data often displayed by CYP3A4. The extreme flexibility revealed by the present structures also challenges any attempt to apply computational design tools without the support of relevant experimental data.

| S-EPMC1564212 | biostudies-literature

Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency.

Project description:NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

| S-EPMC3158178 | biostudies-literature

Structural Basis of Single-Nucleotide Polymorphisms in Cytochrome P450 2C9.

Project description:Single-nucleotide polymorphisms in drug-metabolizing cytochrome P450 (CYP) enzymes are important contributors to interindividual differences in drug metabolism leading to adverse drug reactions. Despite their extensive characterization and importance in pharmacogenetics of clinical drugs, the structural basis of CYP polymorphisms has remained scant. Here we report the crystal structures of human CYP2C9 and its polymorphic variants, *3 (I359L) and *30 (A477T), with an antihypertensive drug losartan. The structures show distinct interaction and occupation of losartan in the active site, the access channel, and the peripheral binding site. The I359L substitution located far from the active site remarkably altered the residue side chains near the active site and the access channel, whereas the T477 substitution illustrated hydrogen-bonding interaction with the reoriented side chain of Q214. The results yield structural insights into the reduced catalytic activity of the CYP2C9 variants and have important implications for understanding genetic polymorphisms in CYP-mediated drug metabolism.

| S-EPMC5761073 | biostudies-literature

Linking cytochrome P450 enzymes from Mycobacterium tuberculosis to their cognate ferredoxin partners.

Project description:Mycobacterium tuberculosis (Mtb) codes for 20 cytochrome P450 enzymes (CYPs), considered potential drug-targets due to their essential roles in bacterial viability and host infection. Catalytic activity of mycobacterial CYPs is dependent on electron transfer from a NAD (P)H-ferredoxin-reductase (FNR) and a ferredoxin (Fd). Two FNRs (FdrA and FprA) and five ferredoxins (Fdx, FdxA, FdxC, FdxD, and Rv1786) have been found in the Mtb genome. However, as of yet, the cognate redox partnerships have not been fully established. This is confounded by the fact that heterologous redox partners are routinely used to reconstitute Mtb CYP metabolism. To this end, this study aimed to biochemically characterize and identify cognate redox partnerships for Mtb CYPs. Interestingly, all combinations of FNRs and ferredoxins were active in the reduction of oxidized cytochrome c, but steady-state kinetic assays revealed FdxD as the most efficient redox partner for FdrA, whereas Fdx coupled preferably with FprA. CYP121A1, CYP124A1, CYP125A1, and CYP142A1 metabolism with the cognate redox partners was reconstituted in vitro showing an unanticipated selectivity in the requirement for electron transfer partnership, which did not necessarily correlate with proximity in the genome. This is the first description of microbial P450 metabolism in which multiple ferredoxins are functionally linked to multiple CYPs.

| S-EPMC6208970 | biostudies-literature

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