RESUMEN
Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 Å crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G-ribosome complex.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Ácido Fusídico/farmacología , Factor G de Elongación Peptídica/metabolismo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Secuencia de Aminoácidos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana , Datos de Secuencia Molecular , Factor G de Elongación Peptídica/genética , Unión Proteica/efectos de los fármacos , Subunidades Ribosómicas Grandes Bacterianas/genética , Staphylococcus aureus/metabolismoRESUMEN
The fatty acid synthase type II enzymatic complex of Mycobacterium tuberculosis (FAS-II(Mt)) catalyzes an essential metabolic pathway involved in the biosynthesis of major envelope lipids, mycolic acids. The partner proteins of this singular FAS-II system represent relevant targets for antituberculous drug design. Two heterodimers of the hydratase 2 protein family, HadAB and HadBC, were shown to be involved in the (3R)-hydroxyacyl-ACP dehydration (HAD) step of FAS-II(Mt) cycles. Recently, an additional member of this family, Rv0241c, was proposed to have the same function, based on the heterologous complementation of a HAD mutant of the yeast mitochondrial FAS-II system. In the present work, Rv0241c was able to complement a HAD mutant in the Escherichia coli model but not a dehydratase-isomerase deficient mutant. However, an enzymatic study of the purified protein demonstrated that Rv0241c possesses a broad chain length specificity for the substrate, unlike FAS-II(Mt) enzymes. Most importantly, Rv0241c exhibited a strict dependence on the coenzyme A (CoA) as opposed to AcpM, the natural acyl carrier protein bearing the chains elongated by FAS-II(Mt). The deletion of Rv0241c showed that this gene is not essential to M. tuberculosis survival in vitro. The resulting mutant did not display any change in the mycolic acid profile. This demonstrates that Rv0241c is a trans-2-enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase that does not belong to FAS-II(Mt). The relevance of a heterologous complementation strategy to identifying proteins of such a system is questioned.
Asunto(s)
Acido Graso Sintasa Tipo II/metabolismo , Ácidos Grasos/metabolismo , Mycobacterium tuberculosis/enzimología , Acido Graso Sintasa Tipo II/clasificación , Acido Graso Sintasa Tipo II/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Ácidos Micólicos/metabolismo , Especificidad por SustratoRESUMEN
The Mycobacterium tuberculosis fatty acid synthase type II (FAS-II) system has the unique property of producing unusually long-chain fatty acids involved in the biosynthesis of mycolic acids, key molecules of the tubercle bacillus. The enzyme(s) responsible for dehydration of (3R)-hydroxyacyl-ACP during the elongation cycles of the mycobacterial FAS-II remained unknown. This step is classically catalyzed by FabZ- and FabA-type enzymes in bacteria, but no such proteins are present in mycobacteria. Bioinformatic analyses and an essentiality study allowed the identification of a candidate protein cluster, Rv0635-Rv0636-Rv0637. Its expression in recombinant Escherichia coli strains leads to the formation of two heterodimers, Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC), which also occurs in Mycobacterium smegmatis, as shown by split-Trp assays. Both heterodimers exhibit the enzymatic properties expected for mycobacterial FAS-II dehydratases: a marked specificity for both long-chain (>or=C(12)) and ACP-linked substrates. Furthermore, they function as 3-hydroxyacyl dehydratases when coupled with MabA and InhA enzymes from the M. tuberculosis FAS-II system. HadAB and HadBC are the long-sought (3R)-hydroxyacyl-ACP dehydratases. The correlation between the substrate specificities of these enzymes, the organization of the orthologous gene cluster in different Corynebacterineae, and the structure of their mycolic acids suggests distinct roles for both heterodimers during the elongation process. This work describes bacterial monofunctional (3R)-hydroxyacyl-ACP dehydratases belonging to the hydratase 2 family. Their original structure and the fact that they are essential for M. tuberculosis survival make these enzymes very good candidates for the development of antimycobacterial drugs.
Asunto(s)
Hidroliasas/metabolismo , Mycobacterium tuberculosis/enzimología , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Catálisis , Simulación por Computador , Escherichia coli/genética , Acido Graso Sintasa Tipo II , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Histidina/metabolismo , Hidroliasas/química , Hidroliasas/genética , Cinética , Espectrometría de Masas , Modelos Biológicos , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mycobacterium tuberculosis/genética , Ácidos Micólicos/química , Ácidos Micólicos/metabolismo , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína , Especificidad por SustratoRESUMEN
A large fraction of the Mycobacterium tuberculosis genome codes for proteins of unknown function. We here report the structure of one of these proteins, Rv0130, solved to a resolution of 1.8 å. The Rv0130 monomer features a single hotdog fold composed of a highly curved beta-sheet on top of a long and a short alpha-helix. Two monomers in turn pack to form a double-hotdog-folded homodimer, similar to a large group of enzymes that use thiol esters as substrates. Rv0130 was found to contain a highly conserved R-specific hydratase motif buried deeply between the two monomers. Our biochemical studies show that the protein is able to hydrate a short trans-2-enoyl-coenzyme A moiety with a k(cat) of 1.1 x 10(2) sec(-1). The importance of the side chains of D40 and H45 for hydratase activity is demonstrated by site-directed mutagenesis. In contrast to many hotdog-folded proteins, a proline residue distorts the central helix of Rv0130. This distortion allows the creation of a long, curved tunnel, similar to the substrate-binding channels of long-chain eukaryotic hydratase 2 enzymes.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Mycobacterium tuberculosis/enzimología , Proteínas Bacterianas/metabolismo , Secuencia Conservada , Cristalografía por Rayos X , Hidroliasas/química , Cinética , Estructura Molecular , Mutagénesis Sitio-Dirigida , Conformación ProteicaRESUMEN
The Mycobacterium tuberculosis genome contains about 4000 genes, of which approximately a third code for proteins of unknown function or are classified as conserved hypothetical proteins. We have determined the three-dimensional structure of one of these, the rv0216 gene product, which has been shown to be essential for M. tuberculosis growth in vivo. The structure exhibits the greatest similarity to bacterial and eukaryotic hydratases that catalyse the R-specific hydration of 2-enoyl coenzyme A. However, only part of the catalytic machinery is conserved in Rv0216 and it showed no activity for the substrate crotonyl-CoA. The structure of Rv0216 allows us to assign new functional annotations to a family of seven other M. tuberculosis proteins, a number if which are essential for bacterial survival during infection and growth.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Supervivencia Celular , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/patogenicidad , Pliegue de Proteína , Acilcoenzima A/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Cristalografía por Rayos X , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Homología de Secuencia de Aminoácido , Tuberculosis/microbiologíaRESUMEN
Structural genomics offers a potential route to the discovery of protein function. As part of a structural genomics project focused on the hyperthermophilic crenarchaeon Pyrobaculum aerophilum, a conserved hypothetical protein, PAE2754, has been expressed in Escherichia coli, purified and crystallized. Because of the difficulties of preparing interpretable heavy-atom derivatives with limited resolution and 8-12 molecules in the asymmetric unit, two leucine residues were selected for mutation to methionine. The double mutant L65M/L80M was created, expressed incorporating SeMet and crystallized. The crystals are monoclinic, space group P2(1), with unit-cell parameters a = 56.4, b = 193.3, c = 60.5 A, beta = 94.6 degrees and eight molecules (two tetramers) in the asymmetric unit. The crystals diffract to 2.75 A resolution and are suitable for MAD phasing.
Asunto(s)
Proteínas Bacterianas/química , Cristalización , Pyrobaculum/química , Proteínas Bacterianas/genética , Clonación Molecular , Cristalografía por Rayos X , Metionina , Mutación MissenseRESUMEN
Genome sequencing projects have focused attention on the problem of discovering the functions of protein domains that are widely distributed throughout living species but which are, as yet, largely uncharacterized. One such example is the PIN domain, found in eukaryotes, bacteria, and Archaea, and with suggested roles in signaling, RNase editing, and/or nucleotide binding. The first reported crystal structure of a PIN domain (open reading frame PAE2754, derived from the crenarchaeon, Pyrobaculum aerophilum) has been determined to 2.5 A resolution and is presented here. Mapping conserved residues from a multiple sequence alignment onto the structure identifies a putative active site. The discovery of distant structural homology with several exonucleases, including T4 phage RNase H and flap endonuclease (FEN1), further suggests a likely function for PIN domains as Mg2+-dependent exonucleases, a hypothesis that we have confirmed in vitro. The tetrameric structure of PAE2754, with the active sites inside a tunnel, suggests a mechanism for selective cleavage of single-stranded overhangs or flap structures. These results indicate likely DNA or RNA editing roles for prokaryotic PIN domains, which are strikingly numerous in thermophiles, and in organisms such as Mycobacterium tuberculosis. They also support previous hypotheses that eukaryotic PIN domains participate in RNAi and nonsense-mediated RNA degradation.