RESUMEN
Mycobacterium abscessus, an opportunistic pathogen responsible for pulmonary infections, contains genes predicted to encode two steroid catabolic pathways: a cholesterol catabolic pathway similar to that of Mycobacterium tuberculosis and a 4-androstenedione (4-AD) catabolic pathway. Consistent with this prediction, M. abscessus grew on both steroids. In contrast to M. tuberculosis, Rhodococcus jostii RHA1, and other Actinobacteria, the cholesterol and 4-AD catabolic gene clusters of the M. abscessus complex lack genes encoding HsaD, the meta-cleavage product (MCP) hydrolase. However, M. abscessus ATCC 19977 harbors two hsaD homologs elsewhere in its genome. Only one of the encoded enzymes detectably transformed steroid metabolites. Among tested substrates, HsaDMab and HsaDMtb of M. tuberculosis had highest substrate specificities for MCPs with partially degraded side chains thioesterified with coenzyme A (kcat/KM = 1.9 × 104 and 5.7 × 103 mM-1s-1, respectively). Consistent with a dual role in cholesterol and 4-AD catabolism, HsaDMab also transformed nonthioesterified substrates efficiently, and a ΔhsaD mutant of M. abscessus grew on neither steroid. Interestingly, both steroids prevented growth of the mutant on acetate. The ΔhsaD mutant of M. abscessus excreted cholesterol metabolites with a fully degraded side chain, while the corresponding RHA1 mutant excreted metabolites with partially degraded side chains. Finally, the ΔhsaD mutant was not viable in macrophages. Overall, our data establish that the cholesterol and 4-AD catabolic pathways of M. abscessus are unique in that they converge upstream of where this occurs in characterized steroid-catabolizing bacteria. The data further indicate that cholesterol is a substrate for intracellular bacteria and that cholesterol-dependent toxicity is not strictly dependent on coenzyme A sequestration.
Asunto(s)
Androstenodiona , Colesterol , Mycobacterium abscessus , Androstenodiona/metabolismo , Colesterol/metabolismo , Coenzima A/metabolismo , Humanos , Hidrolasas/metabolismo , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismoRESUMEN
The meta-cleavage product (MCP) hydrolases utilize a Ser-His-Asp triad to hydrolyze a carbon-carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ES(red), which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ES(red) decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2 (ßnuc â¼ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His-Asp pair does not play an essential role. The data further suggest that ES(red) represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.
Asunto(s)
Ácido Aspártico/química , Proteínas Bacterianas/química , Dipéptidos/química , Ácidos Grasos Insaturados/química , Hidrolasas/química , Sphingomonas/enzimología , Acilación , Ácido Aspártico/metabolismo , Proteínas Bacterianas/metabolismo , Catálisis , Cromatografía Liquida , Dipéptidos/metabolismo , Ácidos Grasos Insaturados/metabolismo , Hidrolasas/metabolismo , Hidrólisis , Cinética , Modelos Químicos , Espectrometría de Masa por Ionización de Electrospray , Especificidad por SustratoRESUMEN
DxnB2 and BphD are meta-cleavage product (MCP) hydrolases that catalyze C-C bond hydrolysis of the biphenyl metabolite 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). BphD is a bottleneck in the bacterial degradation of polychlorinated biphenyls (PCBs) by the Bph catabolic pathway due in part to inhibition by 3-Cl HOPDAs. By contrast, DxnB2 from Sphingomonas wittichii RW1 catalyzes the hydrolysis of 3-Cl HOPDAs more efficiently. X-ray crystallographic studies of the catalytically inactive S105A variant of DxnB2 complexed with 3-Cl HOPDA revealed a binding mode in which C1 through C6 of the dienoate are coplanar. The chlorine substituent is accommodated by a hydrophobic pocket that is larger than the homologous site in BphDLB400 from Burkholderia xenovorans LB400. The planar binding mode observed in the crystalline complex was consistent with the hyper- and hypsochromically shifted absorption spectra of 3-Cl and 3,9,11-triCl HOPDA, respectively, bound to S105A in solution. Moreover, ES(red), an intermediate possessing a bathochromically shifted spectrum observed in the turnover of HOPDA, was not detected, suggesting that substrate destabilization was rate-limiting in the turnover of these PCB metabolites. Interestingly, electron density for the first α-helix of the lid domain was poorly defined in the dimeric DxnB2 structures, unlike in the tetrameric BphDLB400. Structural comparison of MCP hydrolases identified the NC-loop, connecting the lid to the α/ß-hydrolase core domain, as a determinant in the oligomeric state and suggests its involvement in catalysis. Finally, an increased mobility of the DxnB2 lid may contribute to the enzyme's ability to hydrolyze PCB metabolites, highlighting how lid architecture contributes to substrate specificity in α/ß-hydrolases.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Hidrolasas/metabolismo , Bifenilos Policlorados/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión/genética , Burkholderia/enzimología , Burkholderia/genética , Cristalografía por Rayos X , Ácidos Grasos Insaturados/química , Hidrolasas/química , Hidrolasas/genética , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Bifenilos Policlorados/química , Multimerización de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Espectrofotometría , Sphingomonas/enzimología , Sphingomonas/genéticaRESUMEN
The SCO protein from the aerobic bacterium Bacillus subtilis (BsSCO) is involved in the assembly of the cytochrome c oxidase complex, and specifically with the Cu(A) center. BsSCO has been proposed to play various roles in Cu(A) assembly including, the direct delivery of copper ions to the Cu(A) site, and/or maintaining the appropriate redox state of the cysteine ligands during formation of Cu(A). BsSCO binds copper in both Cu(II) and Cu(I) redox states, but has a million-fold higher affinity for Cu(II). As a prerequisite to kinetic studies, we measured equilibrium stability of oxidized, reduced and Cu(II)-bound BsSCO by chemical and thermal induced denaturation. Oxidized and reduced apo-BsSCO exhibit two-state behavior in both chemical- and thermal-induced unfolding. However, the Cu(II) complex of BsSCO is stable in up to nine molar urea. Thermal or guanidinium-induced unfolding of BsSCO-Cu(II) ensues only as the Cu(II) species is lost. The effect of copper (II) on the folding of BsSCO is complicated by a rapid redox reaction between copper and reduced, denatured BsSCO. When denatured apo-BsSCO is refolded in the presence of copper (II) some of the population is recovered as the BsSCO-Cu(II) complex and some is oxidized indicating that refolding and oxidation are competing processes. The proposed functional roles for BsSCO in vivo require that its cysteine residues are reduced and the presence of copper during folding may be detrimental to BsSCO attaining its functional state.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Cobre/metabolismo , Cisteína/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Proteínas de la Membrana/metabolismo , Cinética , Ligandos , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Espectrometría de Fluorescencia , Termodinámica , UreaRESUMEN
Rhodococcus is a genus of mycolic-acid-containing actinomycetes that utilize a remarkable variety of organic compounds as growth substrates. This degradation helps maintain the global carbon cycle and has increasing applications ranging from the biodegradation of pollutants to the biocatalytic production of drugs and hormones. We have been using Rhodococcus jostii RHA1 as a model organism to understand the catabolic versatility of Rhodococcus and related bacteria. Our approach is exemplified by the discovery of a cluster of genes specifying the catabolism of cholesterol. This degradation proceeds via ß-oxidative degradation of the side chain and O2-dependent cleavage of steroid ring A in a process similar to bacterial degradation of aromatic compounds. The pathway is widespread in Actinobacteria and is critical to the pathogenesis of Mycobacterium tuberculosis, arguably the world's most successful pathogen. The close similarity of some of these enzymes with biphenyl- and polychlorinated-biphenyl-degrading enzymes that we have characterized is facilitating inhibitor design. Our studies in RHA1 have also provided important insights into a number of novel metalloenzymes and their biosynthesis, such as acetonitrile hydratase (ANHase), a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. Molecular genetic and biochemical studies have identified AnhE as a dimeric metallochaperone that delivers cobalt to ANHase, enabling its maturation in vivo. Other metalloenzymes we are characterizing include N-acetylmuramic acid hydroxylase, which catalyzes an unusual hydroxylation of the rhodococcal and mycobacterial peptidoglycan, and 2 RHA1 dye-decolorizing peroxidases. Using molecular genetic and biochemical approaches, we have demonstrated that one of these enzymes is involved in the degradation of lignin. Overall, our studies are providing fundamental insights into a range of catabolic processes that have a wide variety of applications.
Asunto(s)
Lignina/metabolismo , Nitrilos/metabolismo , Rhodococcus/enzimología , Esteroides/metabolismo , Biodegradación Ambiental , Colesterol/metabolismo , Hidrolasas/genética , Hidrolasas/metabolismo , Peroxidasas/genética , Peroxidasas/metabolismo , Rhodococcus/genéticaRESUMEN
In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5-9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4-33 times higher specificity for DSHA (k(cat)/K(m) = 3.3 +/- 0.3 x 10(4) m(-1) s(-1)) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a K(d) of 51 +/- 2 mum. The S114A.DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8-1.9 Aindicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the alpha-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 A, supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases.
Asunto(s)
Colesterol/metabolismo , Hidrolasas/metabolismo , Mycobacterium tuberculosis/enzimología , Sustitución de Aminoácidos/genética , Biocatálisis , Colesterol/química , Cristalografía por Rayos X , Ácidos Grasos Insaturados/química , Ácidos Grasos Insaturados/metabolismo , Hidrolasas/química , Cinética , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estructura Secundaria de Proteína , Soluciones , Espectrofotometría Ultravioleta , Electricidad Estática , Especificidad por Sustrato , Torsión MecánicaRESUMEN
Mycobacterium tuberculosis, the etiological agent of TB, possesses a cholesterol catabolic pathway implicated in pathogenesis. This pathway includes an iron-dependent extradiol dioxygenase, HsaC, that cleaves catechols. Immuno-compromised mice infected with a DeltahsaC mutant of M. tuberculosis H37Rv survived 50% longer than mice infected with the wild-type strain. In guinea pigs, the mutant disseminated more slowly to the spleen, persisted less successfully in the lung, and caused little pathology. These data establish that, while cholesterol metabolism by M. tuberculosis appears to be most important during the chronic stage of infection, it begins much earlier and may contribute to the pathogen's dissemination within the host. Purified HsaC efficiently cleaved the catecholic cholesterol metabolite, DHSA (3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione; k(cat)/K(m) = 14.4+/-0.5 microM(-1) s(-1)), and was inactivated by a halogenated substrate analogue (partition coefficient<50). Remarkably, cholesterol caused loss of viability in the DeltahsaC mutant, consistent with catechol toxicity. Structures of HsaC:DHSA binary complexes at 2.1 A revealed two catechol-binding modes: bidentate binding to the active site iron, as has been reported in similar enzymes, and, unexpectedly, monodentate binding. The position of the bicyclo-alkanone moiety of DHSA was very similar in the two binding modes, suggesting that this interaction is a determinant in the initial substrate-binding event. These data provide insights into the binding of catechols by extradiol dioxygenases and facilitate inhibitor design.
Asunto(s)
Proteínas Bacterianas/metabolismo , Colesterol/metabolismo , Mycobacterium tuberculosis/patogenicidad , Oxigenasas/química , Oxigenasas/metabolismo , Animales , Proteínas Bacterianas/química , Cristalografía por Rayos X , Femenino , Cobayas , Ratones , Ratones SCID , Mutación , Mycobacterium tuberculosis/metabolismo , Oxigenasas/genética , Reacción en Cadena de la Polimerasa , Relación Estructura-Actividad , Tuberculosis Pulmonar/metabolismo , Tuberculosis Pulmonar/patologíaRESUMEN
Rhodococcus sp. strain RHA1, a soil bacterium related to Mycobacterium tuberculosis, degrades an exceptionally broad range of organic compounds. Transcriptomic analysis of cholesterol-grown RHA1 revealed a catabolic pathway predicted to proceed via 4-androstene-3,17-dione and 3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3,4-DHSA). Inactivation of each of the hsaC, supAB, and mce4 genes in RHA1 substantiated their roles in cholesterol catabolism. Moreover, the hsaC(-) mutant accumulated 3,4-DHSA, indicating that HsaC(RHA1), formerly annotated as a biphenyl-degrading dioxygenase, catalyzes the oxygenolytic cleavage of steroid ring A. Bioinformatic analyses revealed that 51 rhodococcal genes specifically expressed during growth on cholesterol, including all predicted to specify the catabolism of rings A and B, are conserved within an 82-gene cluster in M. tuberculosis H37Rv and Mycobacterium bovis bacillus Calmette-Guérin. M. bovis bacillus Calmette-Guérin grew on cholesterol, and hsaC and kshA were up-regulated under these conditions. Heterologously produced HsaC(H37Rv) and HsaD(H37Rv) transformed 3,4-DHSA and its ring-cleaved product, respectively, with apparent specificities approximately 40-fold higher than for the corresponding biphenyl metabolites. Overall, we annotated 28 RHA1 genes and proposed physiological roles for a similar number of mycobacterial genes. During survival of M. tuberculosis in the macrophage, these genes are specifically expressed, and many appear to be essential. We have delineated a complete suite of genes necessary for microbial steroid degradation, and pathogenic mycobacteria have been shown to catabolize cholesterol. The results suggest that cholesterol metabolism is central to M. tuberculosis's unusual ability to survive in macrophages and provide insights into potential targets for novel therapeutics.
Asunto(s)
Colesterol/genética , Colesterol/metabolismo , Hidrolasas/genética , Macrófagos/microbiología , Familia de Multigenes , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/genética , Rhodococcus/genética , Hidrolasas/metabolismo , Mycobacterium tuberculosis/metabolismo , ARN Mensajero/metabolismo , Rhodococcus/metabolismoRESUMEN
Members of the Sco protein family are implicated in the assembly of the respiratory complex cytochrome c oxidase. Several possible roles have been proposed for Sco: a copper delivery agent, a site-specific thiol reductase, and an indicator of cellular redox status. Two cysteine residues (C45 and C49) in the sequence CXXXCP and a histidine (H135) approximately 90 residues toward the C-terminus are conserved in Sco from bacteria, yeast, and humans. The soluble domain of Sco has a thioredoxin fold that is suggestive of redox activity for this protein. We have characterized the soluble domain of the Sco protein from Bacillus subtilis (i.e., sBsSco) for its redox reactivity and metal binding capacity. In oxidized sBsSco, the cysteines are present as an intramolecular disulfide. Oxidized sBsSco does not bind metal, but can be reduced in vitro to a metal-binding form. Reduction of the disulfide in sBsSco is accompanied by increased intrinsic fluorescence. The reducibility of the cystine is unchanged when the conserved histidine is mutated to alanine. Tight binding by reduced sBsSco is observed for Cu(II) by electronic absorption, intrinsic fluorescence, and EPR spectroscopies, and isothermal titration calorimetry with an observed stoichiometry of one Cu(II) ion per sBsSco and a KD of approximately 50 nM. Tight binding of Cu(I) and Ag(I) is observed by quenching of intrinsic tryptophan fluorescence. Cobalt(II) exhibits weak binding, whereas Ni(II) and Zn(II) do not appear to bind. The high-affinity binding of metals by BsSco is triggered by its redox state, and this property could be important for its function in vivo.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de la Membrana/química , Metales/química , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Calorimetría , Cationes/química , Cationes/metabolismo , Cationes Bivalentes/química , Cationes Monovalentes/química , Cationes Monovalentes/metabolismo , Cobre/química , Cisteína/química , Cistina/química , Disulfuros/química , Ditiotreitol/química , Espectroscopía de Resonancia por Spin del Electrón , Complejo IV de Transporte de Electrones/metabolismo , Cinética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metales/metabolismo , Mutación/genética , Oxidación-Reducción , Fosfinas/química , Unión Proteica , Piridinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Espectrofotometría , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
The high-affinity calcium-mediated type II cohesin-dockerin interaction is responsible for the attachment of the multi-enzyme cellulose-degrading complex, termed the cellulosome, to the cell surface of the thermophilic anaerobe Clostridium thermocellum. A trimodular 40 kDa complex comprising the SdbA type II cohesin and the the CipA type II dockerin-X module modular pair from the cellulosome of C. thermocellum has been crystallized. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.21, b = 52.34, c = 154.69 A. The asymmetric unit contains one molecule of the protein complex and native and selenomethionine-derivative crystals diffracted to 2.1 and 2.0 A, respectively.