Mycobacterium tuberculosis, the causative agent of human tuberculosis, and Mycobacterium bovis 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-Gué rin 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 (trHbN⅐O2) 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 trHbN⅐O2 and NO at 23°C was 745 M ؊1 ⅐s ؊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 trHbNs ability to detoxify NO.

SummaryThe 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 highresolution 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 (Mtb) is an intracellular pathogen that infects 10 million 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.

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.T runcated hemoglobins (trHbs) are a class of small oxygenbinding hemoproteins, dispersed in eubacteria, cyanobacteria, protozoa, and plants, recently recognized as a separate cluster within the hemoglobin (Hb) superfamily. On the basis of amino acid sequence analysis, three phylogenetic groups (groups I, II, and III) have been identified within the trHb family; some organisms contain genes from more than one group, suggesting different functions for trHbs belonging to the diverse groups (1). Crystal structures of three group I trHbs (2, 3) revealed that trHbs are clearly not just another variation on the motif of vertebrate myoglobin (Mb) and Hb. Neither are they similar to nonvertebrate Hbs, including the heme-containing domain of flavohemoglobins, nor to the plant symbiotic and nonsymbiotic Hbs (4-10). Major structural differences associated with known trHbs are an unprecedented 2-on-2 ␣-helical sandwich fold, resulting from striking editing of the classical 3-on-3 globin ␣-helical sandwich, and an extended hydrophobic tunnel͞cavity network linking the solvent space and the distal heme pocket (1-3). Much smaller and topologically unrelated cavities, known by their ability to incorporate Xe atoms, have been found in Mb and interpreted as temporary ligand-docking sites (11,12). In trHbs, the positioning and size of the hydrophobic tunnel suggest important roles in controlling ligand access to the heme, in ligand storage, and͞or accumulation (1-3).An additional major diffe...