Ethionamide (ETH) plays a central role in the treatment of tuberculosis in patients resistant to the first-line drugs. The ETH, thioamide, and thiourea class of antituberculosis agents are prodrugs that are oxidatively converted to their active S-oxides by the mycobacterial flavin-dependent monooxygenase (EtaA) of Mycobacterium tuberculosis, thus initiating the chain of reactions that result in inhibition of mycolic acid biosynthesis and cell lysis. As part of a search for new lead candidates, we report here that several xanthates are oxidized by purified EtaA to S-oxide metabolites (perxanthates), which are implicated in the antimycobacterial activity of these compounds. This process, which is analogous to that responsible for activation of ETH, is also catalyzed by human flavoprotein monooxygenase 3. EtaA was not inhibited in a time-dependent manner during the reaction. Xanthates with longer alkyl chains were oxidized more efficiently. EtaA oxidized octyl-xanthate (Km = 5 µM; Vmax = 1.023 nmolP/min; kcat = 5.2 molP/min/molE) more efficiently than ETH (194 µM; 1.46 nmolP/min; 7.73 nmolP/min/molE, respectively). Furthermore, the in vitro antimycobacterial activity of four xanthates against M. tuberculosis H37Hv was higher (minimum inhibitory concentration of around 1 µM) than that of ETH (12 µM).

Human heme oxygenases 1 and 2 (HO-1 and HO-2) degrade heme in the presence of oxygen and NADPH-cytochrome P450 reductase, producing ferrous iron, CO, and biliverdin. HO-1 is an inducible enzyme, but HO-2 is constitutively expressed in selected tissues and is involved in signaling and regulatory processes. HO-2 has three cysteine residues that have been proposed to modulate the affinity for heme, whereas HO-1 has none. Here we use site-specific mutagenesis and two-dimensional NMR of l-[3-(13)C]cysteine-labeled proteins to determine the redox state of the individual cysteines in HO-2 and assess their roles in binding of heme. The results indicate that in the apoprotein, Cys(282) and Cys(265) are in the oxidized state, probably in an intramolecular disulfide bond. The addition of a reducing agent converts them to the reduced, free thiol state. Two-dimensional NMR of site-specific mutants reveals that the redox state of Cys(265) and Cys(282) varies with the presence or absence of other Cys residues, indicating that the microenvironments of the Cys residues are mutually interdependent. Cys(265) appears to be in a relatively hydrophilic, oxidizable environment compared with Cys(127) and Cys(282). Chemical shift data indicate that none of the cysteines stably coordinates to the heme iron atom. In the oxidized state of the apoprotein, heme is bound 2.5-fold more tightly than in the reduced state. This small difference in heme affinity between the oxidized and reduced states of the protein is much less than previously reported, suggesting that it is not a significant factor in the physiological regulation of cellular heme levels.

Mycobacteria share a common cholesterol degradation pathway initiated by oxidation of the alkyl side chain by enzymes of cytochrome P450 (CYP) families 125 and 142. Structural and sequence comparisons of the two enzyme families revealed two insertions into the N-terminal region of the CYP125 family (residues 58-67 and 100-109 in the CYP125A1 sequence) that could potentially sterically block the oxidation of the longer cholesterol ester molecules. Catalytic assays revealed that only CYP142 enzymes are able to oxidize cholesteryl propionate, and although CYP125 enzymes could oxidize cholesteryl sulfate, they were much less efficient at doing so than the CYP142 enzymes. The crystal structure of CYP142A2 in complex with cholesteryl sulfate revealed a substrate tightly fit into a smaller active site than was previously observed for the complex of CYP125A1 with 4-cholesten-3-one. We propose that the larger CYP125 active site allows for multiple binding modes of cholesteryl sulfate, the majority of which trigger the P450 catalytic cycle, but in an uncoupled mode rather than one that oxidizes the sterol. In contrast, the more unhindered and compact CYP142 structure enables enzymes of this family to readily oxidize cholesteryl esters, thus providing an additional source of carbon for mycobacterial growth.

Ethionamide (ETH) plays a central role in the treatment of tuberculosis in patients resistant to the first-line drugs. The ETH, thioamide, and thiourea class of antituberculosis agents are prodrugs that are oxidatively converted to their active S-oxides by the mycobacterial flavin-dependent monooxygenase (EtaA) of Mycobacterium tuberculosis, thus initiating the chain of reactions that result in inhibition of mycolic acid biosynthesis and cell lysis. As part of a search for new lead candidates, we report here that several xanthates are oxidized by purified EtaA to S-oxide metabolites (perxanthates), which are implicated in the antimycobacterial activity of these compounds. This process, which is analogous to that responsible for activation of ETH, is also catalyzed by human flavoprotein monooxygenase 3. EtaA was not inhibited in a time-dependent manner during the reaction. Xanthates with longer alkyl chains were oxidized more efficiently. EtaA oxidized octyl-xanthate (Km = 5 µM; Vmax = 1.023 nmolP/min; kcat = 5.2 molP/min/molE) more efficiently than ETH (194 µM; 1.46 nmolP/min; 7.73 nmolP/min/molE, respectively). Furthermore, the in vitro antimycobacterial activity of four xanthates against M. tuberculosis H37Hv was higher (minimum inhibitory concentration of around 1 µM) than that of ETH (12 µM).

Cholest-4-en-3-one, whether added exogenously or generated intracellularly from cholesterol, inhibits the growth ofMycobacterium tuberculosiswhen CYP125A1 and CYP142A1, the cytochrome P450 enzymes that initiate degradation of the sterol side chain, are disabled. Here we demonstrate that a 16-hydroxy derivative of cholesterol, which was previously reported to inhibit growth ofM. tuberculosis, acts by preventing the oxidation of the sterol side chain even in the presence of the relevant cytochrome P450 enzymes. The finding that (25R)-cholest-5-en-3β,16β,26-triol (1) (and its 3-keto metabolite) inhibit growth suggests that cholesterol analogs with non-degradable side chains represent a novel class of anti-mycobacterial agents. In accord with this, two cholesterol analogs with truncated, fluorinated side chains have been synthesized and shown to similarly block the growth in culture ofM. tuberculosis.

Mycobacterium tuberculosis DosS is critical for the induction of M. tuberculosis dormancy genes in response to nitric oxide (NO), carbon monoxide (CO), or hypoxia. These environmental stimuli, which are sensed by the DosS heme group, result in autophosphorylation of a DosS His residue, followed by phosphotransfer to an Asp residue of the response regulator DosR. To clarify the mechanism of gaseous ligand recognition and signaling, we investigated the hydrogen-bonding interactions of the iron-bound CO and NO ligands by site-directed mutagenesis of Glu-87 and His-89. Autophosphorylation assays and molecular dynamics simulations suggest that Glu-87 has an important role in ligand recognition, whereas His-89 is essential for signal transduction to the kinase domain, a process for which Arg-204 is important. Mutation of Glu-87 to Ala or Gly rendered the protein constitutively active as a kinase, but with lower autophosphorylation activity than the wild-type in the Fe(II) and the Fe(II)-CO states, whereas the E87D mutant had little kinase activity except for the Fe(II)-NO complex. The H89R mutant exhibited attenuated autophosphorylation activity, although the H89A and R204A mutants were inactive as kinases, emphasizing the importance of these residues in communication to the kinase core. Resonance Raman spectroscopy of the wild-type and H89A mutant indicates the mutation does not alter the heme coordination number, spin state, or porphyrin deformation state, but it suggests that interdomain interactions are disrupted by the mutation. Overall, these results confirm the importance of the distal hydrogen-bonding network in ligand recognition and communication to the kinase domain and reveal the sensitivity of the system to subtle differences in the binding of gaseous ligands.

The heme-containing cytochrome P450s exhibit isoform-dependent ferric spin equilibria in the resting state and differential substrate-dependent spin equilibria. The basis for these differences is not well understood. Here, magnetic circular dichroism (MCD) reveals significant differences in the resting low spin ligand field of CYPs 3A4, 2E1, 2C9, 125A1, and 51B1, which indicates differences in the strength of axial water ligation to the heme. The near-infrared bands that specifically correspond to charge-transfer porphyrin-to-metal transitions span a range of energies of nearly 2 kcal/mol. In addition, the experimentally determined MCD bands are not entirely in agreement with the expected MCD energies calculated from electron paramagnetic resonance parameters, thus emphasizing the need for the experimental data. MCD marker bands of the high spin heme between 500 and 680 nm were also measured and suggest only a narrow range of energies for this ensemble of high spin Cys(S(-)) → Fe(3+) transitions among these isoforms. The differences in axial ligand energies between CYP isoforms of the low spin states likely contribute to the energetics of substrate-dependent spin state perturbation. However, these ligand field energies do not correlate with the fraction of high spin vs low spin in the resting state enzyme, suggestive of differences in water access to the heme or isoform-dependent differences in the substrate-free high spin states as well.

The heme-containing cytochrome P450s exhibit isoform-dependent ferric spin equilibria in the resting state and differential substrate-dependent spin equilibria. The basis for these differences is not well understood. Here, magnetic circular dichroism (MCD)