RESUMEN
Enzymes orchestrating methylation between tetrahydrofolate (THF) and cobalamin (Cbl) are abundant among all domains of life. During energy production in Desulfitobacterium hafniense, MtgA catalyzes the methyl transfer from methylcobalamin (Cbl-CH3 ) to THF in the catabolism of glycine betaine (GB). Despite its lack of sequence identity with known structures, we could show that MtgA forms a homodimeric complex of two TIM barrels. Atomic crystallographic insights into the interplay of MtgA with THF as well as analysis of a trapped reaction intermediate (THF-CH3 )+ reveal conformational rearrangements during the transfer reaction. Whereas residues for THF methylation are conserved, the binding mode for the THF glutamyl-p-aminobenzoate moiety (THF tail) is unique. Apart from snapshots of individual reaction steps of MtgA, structure-based mutagenesis combined with enzymatic activity assays allowed a mechanistic description of the methyl transfer between Cbl-CH3 and THF. Altogether, the THF-tail-binding motion observed in MtgA is unique compared to other THF methyltransferases and therefore contributes to the general understanding of THF-mediated methyl transfer.
Asunto(s)
Betaína/metabolismo , Desulfitobacterium/química , Tetrahidrofolatos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Betaína/química , Biocatálisis , Cristalografía por Rayos X , Desulfitobacterium/metabolismo , Metilación , Modelos Moleculares , Estructura Molecular , Tetrahidrofolatos/químicaRESUMEN
Sulfation is an important way for detoxifying xenobiotics and endobiotics including catechols. Enzymatic sulfation occurs usually with high chemo- and/or regioselectivity under mild reaction conditions. In this study, a two-step p-NPS-4-AAP screening system for laboratory evolution of aryl sulfotransferase B (ASTB) was developed in 96-well microtiter plates to improve the sulfate transfer efficiency toward catechols. Increased transfer efficiency and improved sulfation stoichiometry are achieved through the two-step screening procedure in a one-pot reaction. In the first step, the p-NPS assay is used (detection of the colorimetric by-product, p-nitrophenol) to determine the apparent ASTB activity. The sulfated product, 3-chlorocatechol-1-monosulfate, is quantified by the 4-aminoantipyrine (4-AAP) assay in the second step. Comparison of product formation to p-NPS consumption ensures successful directed evolution campaigns of ASTB. Optimization yielded a coefficient of variation below 15% for the two-step screening system (p-NPS-4-AAP). In total, 1760 clones from an ASTB-SeSaM library were screened toward the improved sulfation activity of 3-chlorocatechol. The turnover number (kcat = 41 ± 2 s-1) and catalytic efficiency (kcat/KM = 0.41 µM-1 s-1) of the final variant ASTB-M5 were improved 2.4- and 2.3-fold compared with ASTB-WT. HPLC analysis confirmed the improved sulfate stoichiometry of ASTB-M5 with a conversion of 58% (ASTB-WT 29%; two-fold improvement). Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) confirmed the chemo- and regioselectivity, which yielded exclusively 3-chlorocatechol-1-monosulfate. For all five additionally investigated catechols, the variant ASTB-M5 achieved an improved kcat value of up to 4.5-fold and sulfate transfer efficiency was also increased (up to 2.3-fold).
Asunto(s)
Arilsulfotransferasa/genética , Proteínas Bacterianas/genética , Catecoles/metabolismo , Desulfitobacterium/enzimología , Sulfatos/metabolismo , Ampirona/química , Ampirona/metabolismo , Arilsulfotransferasa/química , Arilsulfotransferasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Catecoles/química , Desulfitobacterium/química , Desulfitobacterium/genética , Evolución Molecular Dirigida , Cinética , Espectroscopía de Resonancia Magnética , Especificidad por Sustrato , Sulfatos/químicaRESUMEN
The Desulfitobacterium genus comprises anaerobic Gram-positive bacteria, of which the majority are facultative organohalide respirers. We here present the genomes of eight strains of Desulfitobacterium spp., including five strains of Desulfitobacterium hafniense, one strain each from D. dichloroeliminans and D. metallireducens, and one strain that had not been assigned to any species prior to this study. The newly sequenced genomes were compared with four previously published desulfitobacterial genomes. The average genome sizes are 5.5, 4.3 and 3.4 Mbp for D. hafniense, D. dehalogenans and D. dichloroeliminans/metallireducens, respectively. The genomes encode up to seven reductive dehalogenases, the genomes of both D. hafniense DP7 and D. metallireducens 853-15AT did not encode any reductive dehalogenase. The latter result was a surprise as D. metallireducens 853-15AT has been reported to carry out organohalide respiration. Unlike reported for the pceABCT gene cluster, the other reductive dehalogenase gene clusters do not show any signs of being genetically mobile. All analyzed desulfitobacterial genomes encode a complete cobalamin synthesis pathway. A menaquinone synthesis pathway was found in all strains except D. dichloroeliminans DCA1T. The detailed analysis of the genome sequence of 12 desulfitobacteria from four different species confirmed that this genus has an extremely large metabolic repertoire.
Asunto(s)
Desulfitobacterium/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Desulfitobacterium/química , Desulfitobacterium/clasificación , Tamaño del Genoma , Genoma Bacteriano , GenómicaRESUMEN
Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1(T) consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA.
Asunto(s)
Desulfitobacterium/genética , Desulfitobacterium/metabolismo , Genómica , Halógenos/metabolismo , Proteómica , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Desulfitobacterium/química , Desulfitobacterium/enzimología , Transporte de Electrón , Formiatos/metabolismo , Fumaratos/metabolismo , Genoma Bacteriano , Datos de Secuencia Molecular , OperónRESUMEN
This study describes the identification and the structural and spectroscopic analysis of a cobalamin-binding protein (termed CobDH) implicated in O-demethylation by the organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2. The 1.5â Å resolution crystal structure of CobDH is presented in the cobalamin-bound state and reveals that the protein is composed of an N-terminal helix-bundle domain and a C-terminal Rossmann-fold domain, with the cobalamin coordinated in the base-off/His-on conformation similar to other cobalamin-binding domains that catalyse methyl-transfer reactions. EPR spectroscopy of CobDH confirms cobalamin binding and reveals the presence of a cob(III)alamin superoxide, indicating binding of oxygen to the fully oxidized cofactor. These data provide the first structural insights into the methyltransferase reactions that occur during O-demethylation by D. hafniense.
Asunto(s)
Desulfitobacterium/química , Transcobalaminas/química , Transcobalaminas/metabolismo , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Desulfitobacterium/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Datos de Secuencia Molecular , Oxidorreductasas O-Demetilantes/química , Oxidorreductasas O-Demetilantes/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Espectrofotometría Ultravioleta , Transcobalaminas/genética , Vitamina B 12/metabolismoRESUMEN
Protein domain family YabP (PF07873) is a family of small protein domains that are conserved in a wide range of bacteria and involved in spore coat assembly during the process of sporulation. The 62-residue fragment of Dsy0195 from Desulfitobacterium hafniense, which belongs to the YabP family, exists as a homodimer in solution under the conditions used for structure determination using NMR spectroscopy. The structure of the Dsy0195 homodimer contains two identical 62-residue monomeric subunits, each consisting of five anti-parallel beta strands (ß1, 23-29; ß2, 31-38; ß3, 41-46; ß4, 49-59; ß5, 69-80). The tertiary structure of the Dsy0195 monomer adopts a cylindrical fold composed of two beta sheets. The two monomer subunits fold into a homodimer about a single C2 symmetry axis, with the interface composed of two anti-parallel beta strands, ß1-ß1' and ß5b-ß5b', where ß5b refers to the C-terminal half of the bent ß5 strand, without any domain swapping. Potential functional regions of the Dsy0195 structure were predicted based on conserved sequence analysis. The Dsy0195 structure reported here is the first representative structure from the YabP family.
Asunto(s)
Proteínas Bacterianas/química , Desulfitobacterium/química , Esporas Bacterianas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Desulfitobacterium/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Desulfitobacterium spp. are ubiquitous organisms with a broad metabolic versatility, and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed. Soluble and membrane-associated proteins obtained from cells of Desulfitobacterium hafniense strain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways, such as PspA, GroEL, and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the Wood-Ljungdahl pathway and electron transfer flavoproteins. These proteomic results suggest that D. hafniense strain TCE1 adapts its physiology to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration.
Asunto(s)
Adaptación Fisiológica , Desulfitobacterium/fisiología , Tetracloroetileno/metabolismo , Proteínas Bacterianas/análisis , Carbono/metabolismo , Desulfitobacterium/química , Desulfitobacterium/metabolismo , Electroforesis en Gel Bidimensional , Metabolismo Energético , Fumaratos/metabolismo , Hidrógeno/metabolismo , Ácido Láctico/metabolismo , Espectrometría de Masas , Redes y Vías Metabólicas/genética , Oxidación-Reducción , Proteoma/análisisRESUMEN
YbbR domains are widespread throughout Eubacteria and are expressed as monomeric units, linked in tandem repeats or cotranslated with other domains. Although the precise role of these domains remains undefined, the location of the multiple YbbR domain-encoding ybbR gene in the Bacillus subtilis glmM operon and its previous identification as a substrate for a surfactin-type phosphopantetheinyl transferase suggests a role in cell growth, division, and virulence. To further characterize the YbbR domains, structures of two of the four domains (I and IV) from the YbbR-like protein of Desulfitobacterium hafniense Y51 were solved by solution nuclear magnetic resonance and X-ray crystallography. The structures show the domains to have nearly identical topologies despite a low amino acid identity (23%). The topology is dominated by ß-strands, roughly following a "figure 8" pattern with some strands coiling around the domain perimeter and others crossing the center. A similar topology is found in the C-terminal domain of two stress-responsive bacterial ribosomal proteins, TL5 and L25. Based on these models, a structurally guided amino acid alignment identifies features of the YbbR domains that are not evident from naïve amino acid sequence alignments. A structurally conserved cis-proline (cis-Pro) residue was identified in both domains, though the local structure in the immediate vicinities surrounding this residue differed between the two models. The conservation and location of this cis-Pro, plus anchoring Val residues, suggest this motif may be significant to protein function.
Asunto(s)
Proteínas Bacterianas/química , Simulación de Dinámica Molecular , Secuencia de Aminoácidos , Cristalografía por Rayos X , Desulfitobacterium/química , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Operón , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The crystal structure of Dhaf4260 from Desulfitobacterium hafniense DCB-2 was determined by single-wavelength anomalous diffraction (SAD) to a resolution of 2.01â Å using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). This protein structure is the first representative of the PF04016 (DUF364) Pfam family and reveals a novel combination of two well known domains (an enolase N-terminal-like fold followed by a Rossmann-like domain). Structural and bioinformatic analyses reveal partial similarities to Rossmann-like methyltransferases, with residues from the enolase-like fold combining to form a unique active site that is likely to be involved in the condensation or hydrolysis of molecules implicated in the synthesis of flavins, pterins or other siderophores. The genome context of Dhaf4260 and homologs additionally supports a role in heavy-metal chelation.
Asunto(s)
Proteínas Bacterianas/química , Desulfitobacterium/química , Metales Pesados/química , Fosfopiruvato Hidratasa/química , Pliegue de Proteína , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Desulfitobacterium/metabolismo , Metales Pesados/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
There is a general need to develop more powerful and more robust methods for structural characterization of homodimers, homo-oligomers, and multiprotein complexes using solution-state NMR methods. In recent years, there has been increasing emphasis on integrating distinct and complementary methodologies for structure determination of multiprotein complexes. One approach not yet widely used is to obtain intermediate and long-range distance constraints from paramagnetic relaxation enhancements (PRE) and electron paramagnetic resonance (EPR)-based techniques such as double electron electron resonance (DEER), which, when used together, can provide supplemental distance constraints spanning to 10-70 A. In this Communication, we describe integration of PRE and DEER data with conventional solution-state nuclear magnetic resonance (NMR) methods for structure determination of Dsy0195, a homodimer (62 amino acids per monomer) from Desulfitobacterium hafniense. Our results indicate that combination of conventional NMR restraints with only one or a few DEER distance constraints and a small number of PRE constraints is sufficient for the automatic NMR-based structure determination program CYANA to build a network of interchain nuclear Overhauser effect constraints that can be used to accurately define both the homodimer interface and the global homodimer structure. The use of DEER distances as a source of supplemental constraints as described here has virtually no upper molecular weight limit, and utilization of the PRE constraints is limited only by the ability to make accurate assignments of the protein amide proton and nitrogen chemical shifts.
Asunto(s)
Proteínas Bacterianas/química , Desulfitobacterium/química , Dimerización , Espectroscopía de Resonancia por Spin del Electrón , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación ProteicaRESUMEN
Certain bacteria are able to conserve energy via the reductive dehalogenation of halo-organic compounds in a respiration-type metabolism. The transcriptional regulator CprK from Desulfitobacterium spp. induces expression of halorespiratory genes upon binding of o-chlorophenol ligands and is reversibly inactivated by oxygen through disulphide bond formation. We report crystal structures of D. hafniense CprK in the ligand-free (both oxidation states), ligand-bound (reduced) and DNA-bound states, making it the first member of the widespread CRP-FNR superfamily for which a complete structural description of both redox-dependent and allosteric molecular rearrangements is available. In conjunction with kinetic and thermodynamic ligand binding studies, we provide a model for the allosteric mechanisms underpinning transcriptional control. Amino acids that play a key role in this mechanism are not conserved in functionally distinct CRP-FNR members. This suggests that, despite significant structural homology, distinct allosteric mechanisms are used, enabling this protein family to control a very wide range of processes.