Hugues Ouellet

Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide

Mycobacterium tuberculosis, the causative agent of human tuberculosis, and Mycobacteriumbovis each express two genes, glbN and glbO, encoding distantly related truncated hemoglobins (trHbs), trHbN and trHbO, respectively. Here we report that disruption of M. bovis bacillus Calmette–Guérin glbN caused a dramatic reduction in the NO-consuming activity of stationary phase cells, and that activity could be restored fully by complementing knockout cells with glbN. Aerobic respiration of knockout cells was inhibited markedly by NO in comparison to that of wild-type cells, indicating a protective function for trHbN. TyrB10, which is highly conserved in trHbs and interacts with the bound oxygen, was found essential for NO consumption. Titration of oxygenated trHbN (trHbN⋅O2) with NO resulted in stoichiometric oxidation of the protein with nitrate as the major product of the reaction. The second-order rate constant for the reaction between trHbN⋅O2 and NO at 23°C was 745 μM−1⋅s−1, demonstrating that trHbN detoxifies NO 20-fold more rapidly than myoglobin. These results establish a role for a trHb and demonstrate an NO-metabolizing activity in M. tuberculosis or M. bovis. trHbN thus might play an important role in persistence of mycobacterial infection by virtue of trHbN′s ability to detoxify NO.

A TyrCD1/TrpG8 hydrogen bond network and a TyrB10-TyrCD1 covalent link shape the heme distal site of Mycobacterium tuberculosis hemoglobin O

Truncated hemoglobins (Hbs) are small hemoproteins, identified in microorganisms and in some plants, forming a separate cluster within the Hb superfamily. Two distantly related truncated Hbs, trHbN and trHbO, are expressed at different developmental stages in Mycobacterium tuberculosis. Sequence analysis shows that the two proteins share 18% amino acid identities and belong to different groups within the truncated Hb cluster. Although a specific defense role against nitrosative stress has been ascribed to trHbN (expressed during the Mycobacterium stationary phase), no clear functions have been recognized for trHbO, which is expressed throughout the Mycobacterium growth phase. The 2.1-Å crystal structure of M. tuberculosis cyano-met trHbO shows that the protein assembles in a compact dodecamer. Six of the dodecamer subunits are characterized by a double conformation for their CD regions and, most notably, by a covalent bond linking the phenolic O atom of TyrB10 to the aromatic ring of TyrCD1, in the heme distal cavity. All 12 subunits display a cyanide ion bound to the heme Fe atom, stabilized by a tight hydrogen-bonded network based on the (globin very rare) TyrCD1 and TrpG8 residues. The small apolar AlaE7 residue leaves room for ligand access to the heme distal site through the conventional “E7 path,” as proposed for myoglobin. Different from trHbN, where a 20-Å protein matrix tunnel is held to sustain ligand diffusion to an otherwise inaccessible heme distal site, the topologically related region in trHbO hosts two protein matrix cavities.

Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects 10 million people worldwide and kills 2 million people every year. The uptake and utilization of nutrients by Mtb within the host cell is still poorly understood, although lipids play an important role in Mtb persistence. The recent identification of a large regulon of cholesterol catabolic genes suggests that Mtb can use host sterol for infection and persistence. In this review, we report on recent progress in elucidation of the Mtb cholesterol catabolic reactions and their potential utility as targets for tuberculosis therapeutic agents.

Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies intracellularly generated cholest-4-en-3-one

The infectivity and persistence of Mycobacterium tuberculosis requires the utilization of host cell cholesterol. We have examined the specific role of cytochrome P450 CYP125A1 in the cholesterol degradation pathway using genetic, biochemical and high‐resolution mass spectrometric approaches. The analysis of lipid profiles from cells grown on cholesterol revealed that CYP125A1 is required to incorporate the cholesterol side‐chain carbon atoms into cellular lipids, as evidenced by an increase in the mass of the methyl‐branched phthiocerol dimycocerosates. We observed that cholesterol‐exposed cells lacking CYP125A1 accumulate cholest‐4‐en‐3‐one, suggesting that this is a physiological substrate for this enzyme. Reconstitution of enzymatic activity with spinach ferredoxin and ferredoxin reductase revealed that recombinant CYP125A1 indeed binds both cholest‐4‐en‐3‐one and cholesterol, efficiently hydroxylates both of them at C‐27, and then further oxidizes 27‐hydroxycholest‐4‐en‐3‐one to cholest‐4‐en‐3‐one‐27‐oic acid. We determined the X‐ray structure of cholest‐4‐en‐3‐one‐bound CYP125A1 at a resolution of 1.58 Å. CYP125A1 is essential for growth of CDC1551 in media containing cholesterol or cholest‐4‐en‐3‐one. In its absence, the latter compound is toxic for both CDC1551 and H37Rv when added with glycerol as a second carbon source. CYP125A1 is a key enzyme in cholesterol metabolism and plays a crucial role in circumventing the deleterious effect of cholest‐4‐en‐3‐one.

MYCOBACTERIUM TUBERCULOSIS CYP130: CRYSTAL STRUCTURE, BIOPHYSICAL CHARACTERIZATION, AND INTERACTIONS WITH ANTIFUNGAL AZOLE DRUGS

CYP130 is one of the 20 Mycobacterium tuberculosis cytochrome P450 enzymes, only two of which, CYP51 and CYP121, have so far been studied as individually expressed proteins. Here we characterize a third heterologously expressed M. tuberculosis cytochrome P450, CYP130, by UV-visible spectroscopy, isothermal titration calorimetry, and x-ray crystallography, including determination of the crystal structures of ligand-free and econazole-bound CYP130 at a resolution of 1.46 and 3.0Å, respectively. Ligand-free CYP130 crystallizes in an “open” conformation as a monomer, whereas the econazole-bound form crystallizes in a “closed” conformation as a dimer. Conformational changes enabling the “open-closed” transition involve repositioning of the BC-loop and the F and G helices that envelop the inhibitor in the binding site and reshape the protein surface. Crystal structure analysis shows that the portion of the BC-loop relocates as much as 18Å between the open and closed conformations. Binding of econazole to CYP130 involves a conformational change and is mediated by both a set of hydrophobic interactions with amino acid residues in the active site and coordination of the heme iron. CYP130 also binds miconazole with virtually the same binding affinity as econazole and clotrimazole and ketoconazole with somewhat lower affinities, which makes it a plausible target for this class of therapeutic drugs. Overall, binding of the azole inhibitors is a sequential two-step, entropy-driven endothermic process. Binding of econazole and clotrimazole exhibits positive cooperativity that may reflect a propensity of CYP130 to associate into a dimeric structure.

Characterization of the LI818 polypeptide from the green unicellular alga Chlamydomonas reinhardtii.

The LI818 gene from Chlamydomonas encodes a polypeptide that is related to the chlorophyll a/b-binding proteins (CAB) of higher plants and green algae. However, despite this relatedness, LI818 gene expression is not coordinated with that of cab genes and is regulated differently by light, suggesting a different role for LI818 polypeptide. We show here that, in contrast to CAB polypeptides, LI818 polypeptide is not tightly embedded into the thylakoid membranes and is localized in stroma-exposed regions. Moreover, during chloroplast development, LI818 polypeptide accumulates before CAB polypeptides. We also show that the LI818 polypeptide forms with certain chlorophyll a/c-binding proteins (CAC) from the haptophyte Isochrysis galbana and the diatom Cyclotella cryptica a natural group that is distinct from those constituted by CAB, CAC and the chlorophyll a/a-binding proteins (CAA). Such an association suggests a very ancient origin for this group of polypeptides, which predates the division of the early photosynthetic eukaryotes into green (chlorophyte), red (rhodophyte) and brown (chromophyte) algae. Possible roles for the LI818 polypeptide are discussed.

Small-Molecule Scaffolds for CYP51 Inhibitors Identified by High-Throughput Screening and Defined by X-Ray Crystallography

ABSTRACT Sterol 14α-demethylase (CYP51), a major checkpoint in membrane sterol biosynthesis, is a key target for fungal antibiotic therapy. We sought small organic molecules for lead candidate CYP51 inhibitors. The changes in CYP51 spectral properties following ligand binding make CYP51 a convenient target for high-throughput screening technologies. These changes are characteristic of either substrate binding (type I) or inhibitor binding (type II) in the active site. We screened a library of 20,000 organic molecules against Mycobacterium tuberculosis CYP51 (CYP51Mt), examined the top type I and type II binding hits for their inhibitory effects on M. tuberculosis in broth culture, and analyzed them spectrally for their ability to discriminate between CYP51Mt and two reference M. tuberculosis CYP proteins, CYP130 and CYP125. We determined the binding mode for one of the top type II hits, α-ethyl-N-4-pyridinyl-benzeneacetamide (EPBA), by solving the X-ray structure of the CYP51Mt-EPBA complex to a resolution of 1.53 Å. EPBA binds coordinately to the heme iron in the CYP51Mt active site through a lone pair of nitrogen electrons and also through hydrogen bonds with residues H259 and Y76, which are invariable in the CYP51 family, and hydrophobic interactions in a phylum- and/or substrate-specific cavity of CYP51. We also identified a second compound with structural and binding properties similar to those of EPBA, 2-(benzo[d]-2,1,3-thiadiazole-4-sulfonyl)-2-amino-2-phenyl-N-(pyridinyl-4)-acetamide (BSPPA). The congruence between the geometries of EPBA and BSPPA and the CYP51 binding site singles out EPBA and BSPPA as lead candidate CYP51 inhibitors with optimization potential for efficient discrimination between host and pathogen enzymes.

Functional Redundancy of Steroid C26-monooxygenase Activity in Mycobacterium tuberculosis Revealed by Biochemical and Genetic Analyses

One challenge to the development of new antitubercular drugs is the existence of multiple virulent strains that differ genetically. We and others have recently demonstrated that CYP125A1 is a steroid C26-monooxygenase that plays a key role in cholesterol catabolism in Mycobacterium tuberculosis CDC1551 but, unexpectedly, not in the M. tuberculosis H37Rv strain. This discrepancy suggests that the H37Rv strain possesses compensatory activities. Here, we examined the roles in cholesterol metabolism of two other cytochrome P450 enzymes, CYP124A1 and CYP142A1. In vitro analysis, including comparisons of the binding affinities and catalytic efficiencies, demonstrated that CYP142A1, but not CYP124A1, can support the growth of H37Rv cells on cholesterol in the absence of cyp125A1. All three enzymes can oxidize the sterol side chain to the carboxylic acid state by sequential oxidation to the alcohol, aldehyde, and acid. Interestingly, CYP125A1 generates oxidized sterols of the (25S)-26-hydroxy configuration, whereas the opposite 25R stereochemistry is obtained with CYP124A1 and CYP142A1. Western blot analysis indicated that CYP124A1 was not detectably expressed in either the H37Rv or CDC1551 strains, whereas CYP142A1 was found in H37Rv but not CDC1551. Genetic complementation of CDC1551 Δcyp125A1 cells with the cyp124A1 or cyp142A1 genes revealed that the latter can fully rescue the growth defect on cholesterol, whereas cells overexpressing CYP124A1 grow poorly and accumulate cholest-4-en-3-one. Our data clearly establish a functional redundancy in the essential C26-monooxygenase activity of M. tuberculosis and validate CYP125A1 and CYP142A1 as possible drug targets.

The Mycobacterium tuberculosis cytochrome P450 system

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.

Reaction of Mycobacterium tuberculosis Truncated Hemoglobin O with Hydrogen Peroxide EVIDENCE FOR PEROXIDATIC ACTIVITY AND FORMATION OF PROTEIN-BASED RADICALS

In this work, we investigated the reaction of ferric Mycobacterium tuberculosis truncated hemoglobin O (trHbO) with hydrogen peroxide. Stopped-flow spectrophotometric experiments under single turnover conditions showed that trHbO reacts with H2O2 to give transient intermediate(s), among which is an oxyferryl heme, different from a typical peroxidase Compound I (oxyferryl heme π-cation radical). EPR spectroscopy indicated evidence for both tryptophanyl and tyrosyl radicals, whereas redox titrations demonstrated that the peroxide-treated protein product retains 2 oxidizing eq. We propose that Compound I formed transiently is reduced with concomitant oxidation of Trp(G8) to give the detected oxoferryl heme and a radical on Trp(G8) (detected by EPR of the trHbO Tyr(CD1)Phe mutant). In the wild-type protein, the Trp(G8) radical is in turn reduced rapidly by Tyr(CD1). In a second cycle, Trp(G8) may be reoxidized by the ferryl heme to yield ferric heme and two protein radicals. In turn, these migrate to form tyrosyl radicals on Tyr55 and Tyr115, which lead, in the absence of a reducing substrate, to oligomerization of the protein. Steady-state kinetics in the presence of H2O2 and the one-electron donor 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) indicated that trHbO has peroxidase activity, in accord with the presence of typical peroxidase intermediates. These findings suggest an oxidation/reduction function for trHbO and, by analogy, for other Group II trHbs.