RESUMO
A putative haloalkane dehalogenase has been identified in a marine Rhodobacteraceae and subsequently cloned and over-expressed in Escherichia coli. The enzyme has highest activity towards the substrates 1,6-dichlorohexane, 1-bromooctane, 1,3-dibromopropane and 1-bromohexane. The crystal structures of the enzyme in the native and product bound forms reveal a large hydrophobic active site cavity. A deeper substrate binding pocket defines the enzyme preference towards substrates with longer carbon chains. Arg136 at the bottom of the substrate pocket is positioned to bind the distal halogen group of extended di-halogenated substrates.
Assuntos
Proteínas de Bactérias/química , Hidrolases/química , Rhodobacteraceae/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Cicloexanos/química , Hidrocarbonetos Halogenados/química , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Propano/análogos & derivados , Propano/química , Ligação Proteica , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
The putative L-haloacid dehalogenase gene (DehRhb) from a marine Rhodobacteraceae family was cloned and overexpressed in Escherichia coli. The DehRhb protein was shown to be an L-haloacid dehalogenase with highest activity towards brominated substrates with short carbon chains (≤ C3). The optimal temperature for enzyme activity was 55 °C, and the Vmax and Km were 1.75 µm·min(-1) ·mg(-1) of protein and 6.72 mm, respectively, when using monobromoacetic acid as a substrate. DehRhb showed moderate thermal stability, with a melting temperature of 67 °C. The enzyme demonstrated high tolerance to solvents, as shown by thermal shift experiments and solvent incubation assays. The DehRhb protein was crystallized and structures of the native, reaction intermediate and substrate-bound forms were determined. The active site of DehRhb had significant differences from previously studied L-haloacid dehalogenases. The asparagine and arginine residues shown to be essential for catalytic activity in other L-haloacid dehalogenases are not present in DehRhb. The histidine residue which replaces the asparagine residue in DehRhb was coordinated by a conformationally strained glutamate residue that replaces a conserved glycine. The His/Glu dyad is positioned for deprotonation of the catalytic water which attacks the ester bond in the reaction intermediate. The catalytic water in DehRhb is shifted by ~ 1.5 Å from its position in other L-haloacid dehalogenases. A similar His/Glu or Asp dyad is known to activate the catalytic water in haloalkane dehalogenases. The DehRhb enzyme represents a novel member within the L-haloacid dehalogenase family and it has potential to be used as a commercial biocatalyst.
Assuntos
Hidrolases/química , Hidrolases/metabolismo , Rhodobacteraceae/enzimologia , Sequência de Aminoácidos , Organismos Aquáticos , Arginina/química , Arginina/metabolismo , Asparagina/química , Asparagina/metabolismo , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Estabilidade Enzimática , Escherichia coli/genética , Glicina/química , Glicina/metabolismo , Histidina/química , Histidina/metabolismo , Hidrolases/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solventes , Especificidade por Substrato , Temperatura , ÁguaRESUMO
The nucleotide excision repair (NER) pathway removes bulky lesions such as photoproducts from DNA. In both bacteria and eukarya, lesions located in transcribed strands are repaired significantly faster than those located in non-transcribed strands due to damage signalling by stalled RNA polymerase molecules: a phenomenon known as transcription-coupled repair (TCR). TCR requires a mechanism for coupling the detection of stalled RNA polymerase molecules to the NER pathway, provided in bacteria by the Mfd protein. In the third domain of life, archaea, the pathway of NER is not well defined, there are no Mfd homologues and the existence of TCR has not been investigated. In this report we looked at rates of removal of photoproducts in three different operons of the crenarchaeon Sulfolobus solfataricus following UV irradiation. We found no evidence for significantly faster repair in the transcribed strands of these three operons. The rate of global genome repair in S. solfataricus is relatively rapid, and this may obviate the requirement for a specialized TCR pathway. Significantly faster repair kinetics were observed in the presence of visible light, consistent with the presence of a gene for photolyase in the genome of S. solfataricus.
Assuntos
Reparo do DNA , DNA Arqueal/metabolismo , Sulfolobus solfataricus/fisiologia , Southern Blotting , DNA Arqueal/efeitos da radiação , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Luz , Análise de Sequência com Séries de Oligonucleotídeos , Óperon/genética , RNA Arqueal/análise , RNA Arqueal/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfolobus solfataricus/efeitos da radiação , Transcrição GênicaRESUMO
BACKGROUND: DNA damage leads to cellular responses that include the increased expression of DNA repair genes, repression of DNA replication and alterations in cellular metabolism. Archaeal information processing pathways resemble those in eukaryotes, but archaeal damage response pathways remain poorly understood. RESULTS: We analyzed the transcriptional response to UV irradiation in two related crenarchaea, Sulfolobus solfataricus and Sulfolobus acidocaldarius. Sulfolobus species encounter high levels of DNA damage in nature, as they inhabit high temperature, aerobic environments and are exposed to sunlight. No increase in expression of DNA repair genes following UV irradiation was observed. There was, however, a clear transcriptional response, including repression of DNA replication and chromatin proteins. Differential effects on the expression of the three transcription factor B (tfb) genes hint at a mechanism for the modulation of transcriptional patterns in response to DNA damage. TFB3, which is strongly induced following UV irradiation, competes with TFB1 for binding to RNA polymerase in vitro, and may act as a repressor of transcription or an alternative transcription factor for certain promoters. CONCLUSION: A clear response to DNA damage was observed, with down-regulation of the DNA replication machinery, changes in transcriptional regulatory proteins, and up-regulation of the biosynthetic enzymes for beta-carotene, which has UV protective properties, and proteins that detoxify reactive oxygen species. However, unlike eukaryotes and bacteria, there was no induction of DNA repair proteins in response to DNA damage, probably because these are expressed constitutively to deal with increased damage arising due to high growth temperatures.
Assuntos
Dano ao DNA , Reparo do DNA/genética , Regulação da Expressão Gênica em Archaea/efeitos da radiação , Sulfolobus acidocaldarius/efeitos da radiação , Sulfolobus solfataricus/efeitos da radiação , Raios Ultravioleta , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Western Blotting , Primers do DNA/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Citometria de Fluxo , Imunoprecipitação , Análise em Microsséries , Estresse Oxidativo/efeitos da radiação , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , beta Caroteno/biossínteseRESUMO
Consortia containing a novel coccus-shaped, anaerobic heterotroph together with Pyrobaculum rods were cultivated from geothermal environments in New Zealand. Pure cultures of the cocci were only obtained from one such consortium, despite extensive attempts. Cells of this strain (AQ1.S1T) were regular to irregular cocci in morphology and occasionally formed large aggregates, especially when utilizing polysaccharides such as konjac glucomannan as a carbon source. Strain AQ1.S1T is a hyperthermophile, with an optimal temperature for growth between 92 and 95 degrees C (range 85-98 degrees C), and a moderate acidophile, with optimal growth occurring at pH 6.4 (range 5.4-7.0). Growth was inhibited by the addition of sulphur and NaCl (optimal growth occurred without addition of NaCl) and an electron acceptor was not required. Strain AQ1.S1T utilized starch, trypticase peptone, lactose, glucose, konjac glucomannan, mannose, galactose, maltose, glycogen and beta-cyclodextrin as carbon sources. The G+C content was 52.9 mol%. Based on 16S rRNA gene sequence analysis and physiological features it is proposed that isolate AQ1.S1T (=DSM 17230T=JCM 13409T) represents the type strain of a novel species of a new genus within the Crenarchaeota, Ignisphaera aggregans gen. nov., sp. nov.
Assuntos
Desulfurococcales/classificação , Desulfurococcales/isolamento & purificação , Fontes Termais/microbiologia , Composição de Bases , Metabolismo dos Carboidratos , Adesão Celular , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Desulfurococcales/citologia , Desulfurococcales/fisiologia , Genes de RNAr/genética , Inibidores do Crescimento/farmacologia , Temperatura Alta , Concentração de Íons de Hidrogênio , Mananas/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Contraste de Fase , Dados de Sequência Molecular , Nova Zelândia , Peptonas/metabolismo , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Cloreto de Sódio/farmacologia , Enxofre/farmacologia , Microbiologia da ÁguaRESUMO
Stable carbon isotopes can provide insight into carbon cycling pathways in natural environments. We examined carbon isotope fractionations associated with a hyperthermophilic fermentative bacterium, Thermotoga maritima, and a thermophilic chemolithoautotrophic bacterium Persephonella marina. In T. maritima, phospholipid fatty acids (PLFA) are slightly enriched in 13C relative to biomass (epsilon = 0.1-0.8 per thousand). However, PLFA and biomass are depleted in 13C relative to the substrate glucose by approximately 8 per thousand. In P. marina, PLFA are 1.8-14.5 per thousand enriched in 13C relative to biomass, which suggests that the reversed tricarboxylic acid (TCA) cycle or the 3-hydroxypropionate pathway may be used for CO2 fixation. This is supported by small fractionation between biomass and CO2 (epsilon = -3.8 per thousand to -5.0 per thousand), which is similar to fractionations reported for other organisms using similar CO2 fixation pathways. Identification of the exact pathway will require biochemical assay for specific enzymes associated with the reversed TCA cycle or the 3-hydroxypropionate pathway.