Distal Hydrogen-bonding Interactions in Ligand Sensing and Signaling by Mycobacterium tuberculosis DosS.

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.

Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography.

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional (1)H,(15)N HSQC chemical shift perturbation mapping of (15)N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.

Specificity determinants of CYP1B1 estradiol hydroxylation.

Cytochrome P450 (P450)-catalyzed oxidation of the aromatic ring of estradiol can result in 2- or 4-hydroxylation. Which of these products is formed is biologically important, as the 4-hydroxylated metabolite is carcinogenic, whereas the 2-hydroxylated metabolite is not. Most human P450 enzymes, including CYP1A1 and CYP1A2, exhibit a high preference for estradiol 2-hydroxylation, but human CYP1B1 greatly favors 4-hydroxylation. Here we show that heterologous expression of the human, monkey, dog, rat, and mouse CYP1B1 enzymes yields active proteins that differ in their estradiol hydroxylation specificity. The monkey and dog orthologs, like the human enzyme, preferentially catalyze 4-hydroxylation, but the rat and mouse enzymes favor 2-hydroxylation. Analysis of the CYP1B1 sequences in light of these findings suggested that one residue, Val395 in human CYP1B1, could account for the differential hydroxylation specificities. In fact, mutation of this valine in human CYP1B1 to the leucine present in the rat enzyme produces a human enzyme that has the 2-hydroxylation specificity of the rat enzyme. The converse is true when the leucine in the rat enzyme is mutated to the human valine. The role of CYP1B1 in estradiol carcinogenicity thus depends on the identity of this single amino acid residue.

Expression in Escherichia coli of a cytochrome P450 enzyme with a cobalt protoporphyrin IX prosthetic group.

Unlike many hemoproteins, the prosthetic heme group of most cytochrome P450 enzymes cannot be extracted and replaced by modified heme groups. Here, we describe a procedure for generating a cytochrome P450 enzyme (CYP119) with cobalt protoporphyrin IX as its prosthetic group. This is achieved by expressing the protein in Escherichia coli in iron-limited medium and adding cobalt to the medium at the moment that inducible protein expression is initiated.

Bioactivation of antituberculosis thioamide and thiourea prodrugs by bacterial and mammalian flavin monooxygenases.

The thioamide and thiourea class of antituberculosis agents encompasses prodrugs that are oxidatively converted to their active forms by the flavin monooxygenase EtaA of Mycobacterium tuberculosis. Reactive intermediates produced in the EtaA-catalyzed transformations of ethionamide and prothionamide result in NAD(+)/NADH adducts that inhibit the enoyl CoA reductase InhA, the ultimate target of these drugs. In the case of thiacetazone and isoxyl, EtaA produces electrophilic metabolites that mediate the antibacterial activity of these agents. The oxidation of the thioamide/thiourea drugs by the human flavin monooxygenases yields similar reactive metabolites that contribute to the toxicities associated with these second line antituberculosis drugs.

Efficient hypoxic activation of the anticancer agent AQ4N by CYP2S1 and CYP2W1.

AQ4N [1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione], a prodrug with two dimethylamino N-oxide groups, is converted to the topoisomerase II inhibitor AQ4 [1,4-bis{[2-(dimethylamino)ethyl]amino}-5,8-dihydroxy-anthracene-9,10-dione] by reduction of the N-oxides to dimethylamino substituents. Earlier studies showed that several drug-metabolizing cytochrome P450 (P450) enzymes can catalyze this reductive reaction under hypoxic conditions comparable with those in solid tumors. CYP2S1 and CYP2W1, two extrahepatic P450 enzymes identified from the human genome whose functions are unknown, are expressed in hypoxic tumor cells at much higher levels than in normal tissue. Here, we demonstrate that CYP2S1, contrary to a published report (Mol Pharmacol 76:1031-1043, 2009), is efficiently reduced by NADPH-P450 reductase. Most importantly, both CYP2S1 and CYP2W1 are better catalysts for the reductive activation of AQ4N to AQ4 than all previously examined P450 enzymes. The overexpression of CYP2S1 and CYP2W1 in tumor tissues, together with their high catalytic activities for AQ4N activation, suggests that they may be exploited for the localized activation of anticancer prodrugs.

Human flavin-containing monooxygenase 2.1 catalyzes oxygenation of the antitubercular drugs thiacetazone and ethionamide.

The second-line antitubercular drugs thiacetazone (TAZ) and ethionamide (ETA) are bioactivated by the mycobacterial enzyme EtaA. We report here that human flavin-containing monooxygenase 2.1 (FMO2.1), which is expressed predominantly in the lung, catalyzes oxygenation of TAZ. The metabolites generated, the sulfenic acid, sulfinic acid, and carbodiimide derivatives, are the same as those produced by EtaA and human FMO1 and FMO3. Two of the metabolites, the sulfenic acid and carbodiimide, are known to be harmful to mammalian cells. FMO2.1 also catalyzes oxygenation of ETA, producing the S-oxide. We have developed a novel spectrophotometric assay for TAZ oxygenation. The assay was used to determine kinetic parameters for TAZ oxygenation catalyzed by human FMO1, FMO2.1, and FMO3 and by EtaA. Although the K(M) values for the four enzyme-catalyzed reactions are similar, k(cat) and, consequently, k(cat)/K(M) (the specificity constant) for FMO2.1-catalyzed TAZ oxygenation are much higher than those of FMO1, FMO3, or EtaA. This indicates that FMO2.1 is more effective in catalyzing TAZ oxygenation than are the other three enzymes and thus is likely to contribute substantially to the metabolism of TAZ, decreasing the availability of the prodrug to mycobacteria and producing toxic metabolites. Because of a genetic polymorphism, Europeans and Asians lack FMO2.1. However, in sub-Saharan Africa, a region in which tuberculosis is a major health problem, a substantial proportion of individuals express FMO2.1. Thus, our results may explain some of the observed interindividual differences in response to TAZ and ETA and have implications for the treatment of tuberculosis in sub-Saharan Africa.

Ligand-induced conformational heterogeneity of cytochrome P450 CYP119 identified by 2D NMR spectroscopy with the unnatural amino acid (13)C-p-methoxyphenylalanine.

Conformational dynamics are thought to play an important role in ligand binding and catalysis by cytochrome P450 enzymes, but few techniques exist to examine them in molecular detail. Using a unique isotopic labeling strategy, we have site specifically inserted a (13)C-labeled unnatural amino acid residue, (13)C-p-methoxyphenylalanine (MeOF), into two different locations in the substrate binding region of the thermophilic cytochrome P450 enzyme CYP119. Surprisingly, in both cases the resonance signal from the ligand-free protein is represented by a doublet in the (1)H,(13)C-HSQC spectrum. Upon binding of 4-phenylimidazole, the signals from the initial resonances are reduced in favor of a single new resonance, in the case of the F162MeOF mutant, or two new resonances, in the case of the F153MeOF mutant. This represents the first direct physical evidence for the ligand-dependent existence of multiple P450 conformers simultaneously in solution. This general approach may be used to further illuminate the role that conformational dynamics plays in the complex enzymatic phenomena exhibited by P450 enzymes.

Reductive heme-dependent activation of the n-oxide prodrug AQ4N by nitric oxide synthase.

Anaerobic reduction of anticancer prodrugs is a promising route to achieve targeting and selectivity in anticancer drug design. Most reductive prodrug activations involve simple electron transfer from a flavoprotein and are not amenable to specific targeting. Here, we report that the N-oxide AQ4N is reduced by a nitric oxide synthase. This reduction involves interaction with the heme iron atom in the active site and is thus subject to specific protein constraints.

Crystal structure and properties of CYP231A2 from the thermoacidophilic archaeon Picrophilus torridus.

The crystal structure of a cytochrome P450 from the thermoacidophile Picrophilus torridus, CYP231A2 (PTO1399), has been solved. This structure reveals a wide open substrate access channel. To better understand ligand-induced structural transitions in CYP231A2, protein-ligand interactions were investigated using 4-phenylimidazole. Comparison of the ligand-free and -bound CYP231A2 structures shows conformational changes where the F and G helices swing as a single rigid body about a pivot point at the N-terminal end of the F helix, allowing the F helix region to dip toward the heme, resulting in closer contacts with the ligand. Thermal melting data illustrate that the melting temperature for CYP231A2 increases nearly 10 degrees C upon ligand binding, thus illustrating that the closed conformation is substantially more stable. Furthermore, spectroscopic data indicate that the active site is stable at pH 4.5, although, unusually, the thiolate ligand to the iron can be reversibly protonated. CYP231A2 does not exhibit structural features normally associated with thermophilic proteins such as an increase in salt bridge networks or extensive aromatic clustering. The increase in thermal stability instead is best correlated with the smaller size and shorter loops in CYP231A2 compared to other P450s.

Thermophilic cytochrome P450 enzymes.

Thermophilic cytochrome P450 enzymes are of potential interest from structural, mechanistic, and biotechnological points of view. The structures and properties of two such enzymes, CYP119 and CYP175A1, have been investigated and provide the foundation for future work on thermophilic P450 enzymes.

EpoK, a cytochrome P450 involved in biosynthesis of the anticancer agents epothilones A and B. Substrate-mediated rescue of a P450 enzyme.

The epothilones are a new class of highly promising anticancer agents with a mode of action akin to that of paclitaxel but with distinct advantages over that drug. The principal natural compounds are epothilones A and B, which have an epoxide in the macrocyclic lactone ring, and C and D, which have a double bond instead of the epoxide group. The epoxidation of epothilones C and D to A and B, respectively, is mediated by EpoK, a cytochrome P450 enzyme encoded in the epothilone gene cluster. Here we report high-yield expression of EpoK, characterization of the protein, demonstration that the natural substrate can prevent-and even reverse-denaturation of the protein, identification of ligands and surrogate substrates, development of a high-throughput fluorescence activity assay based on the H(2)O(2)-dependent oxidation of 7-ethoxy-4-trifluoromethylcoumarin, and identification of effective inhibitors of the enzyme. These results will facilitate improvements in the yields of epothilones C and D and the engineering of EpoK to prepare novel epothilone analogues. Furthermore, the finding that the denatured enzyme is rescued by the substrate offers a potential paradigm for control of the P450 catalytic function.

Nitric-oxide synthase (NOS) reductase domain models suggest a new control element in endothelial NOS that attenuates calmodulin-dependent activity.

Inducible (iNOS) and constitutive (eNOS, nNOS) nitric-oxide synthases differ in their Ca2+-calmodulin (CaM) dependence. iNOS binds CaM irreversibly but eNOS and nNOS, which bind CaM reversibly, have inserts in their reductase domains that regulate electron transfer. These include the 43-45-amino acid autoinhibitory element (AI) that attenuates electron transfer in the absence of CaM, and the C-terminal 20-40-amino acid tail that attenuates electron transfer in a CaM-independent manner. We constructed models of the reductase domains of the three NOS isoforms to predict the structural basis for CaM-dependent regulation. We have identified and characterized a loop (CD2A) within the NOS connecting domain that is highly conserved by isoform and that, like the AI element, is within direct interaction distance of the CaM binding region. The eNOS CD2A loop (eCD2A) has the sequence 834KGSPGGPPPG843, and is truncated to 809ESGSY813 (iCD2A) in iNOS. The eCD2A contributes to the Ca2+ dependence of CaM-bound activity to a level similar to that of the AI element. The eCD2A plays an autoinhibitory role in the control of NO, and CaM-dependent and -independent reductase activity, but this autoinhibitory function is masked by the dominant AI element. Finally, the iCD2A is involved in determining the salt dependence of NO activity at a post-flavin reduction level. Electrostatic interactions between the CD2A loop and the CaM-binding region, and CaM itself, provide a structural means for the CD2A to mediate CaM regulation of intra-subunit electron transfer within the active NOS complex.

Electron supply and catalytic oxidation of nitrogen by cytochrome P450 and nitric oxide synthase.

Cytochrome P450 and nitric oxide synthase (NOS) oxidize nitrogen atoms, although the substrates and transformations are highly restricted for NOS. The first reaction catalyzed by NOS is mediated by a P450-like ferryl species, although it is generated by a distinct process in which a tetrahydrobiopterin molecule in NOS serves as a transient electron donor. The second NOS reaction appears to be mediated by an iron dioxygen precursor of the ferryl species. The transient tetrahydrobiopterin radical formed in these reactions is quenched by electron transfer from the NOS flavin domain. Electron transfer from the flavins is controlled by the binding of calmodulin, the presence of peptide inserts in the flavin domain, the substrate structure, and phosphorylation of the enzyme.

The crystal structure of Mycobacterium tuberculosis alkylhydroperoxidase AhpD, a potential target for antitubercular drug design.

The resistance of Mycobacterium tuberculosis to isoniazid is commonly linked to inactivation of a catalase-peroxidase, KatG, that converts isoniazid to its biologically active form. Loss of KatG is associated with elevated expression of the alkylhydroperoxidases AhpC and AhpD. AhpD has no sequence identity with AhpC or other proteins but has alkylhydroperoxidase activity and possibly additional physiological activities. The alkylhydroperoxidase activity, in the absence of KatG, provides an important antioxidant defense. We have determined the M. tuberculosis AhpD structure to a resolution of 1.9 A. The protein is a trimer in a symmetrical cloverleaf arrangement. Each subunit exhibits a new all-helical protein fold in which the two catalytic sulfhydryl groups, Cys-130 and Cys-133, are located near a central cavity in the trimer. The structure supports a mechanism for the alkylhydroperoxidase activity in which Cys-133 is deprotonated by a distant glutamic acid via the relay action of His-137 and a water molecule. The cysteine then reacts with the peroxide to give a sulfenic acid that subsequently forms a disulfide bond with Cys-130. The crystal structure of AhpD identifies a new protein fold relevant to members of this protein family in other organisms. The structural details constitute a potential platform for the design of inhibitors of potential utility as antitubercular agents and suggest that AhpD may have disulfide exchange properties of importance in other areas of M. tuberculosis biology.