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1.
Appl Microbiol Biotechnol ; 103(14): 5727-5737, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31123770

RESUMO

Transaminase enzymes (TAms) are becoming increasingly valuable in the chemist's toolbox as a biocatalytic route to chiral amines. Despite high profile successes, the lack of (R)-selective TAms and robustness under harsh industrial conditions continue to prove problematic. Herein, we report the isolation of the first haloarchaeal TAm (BC61-TAm) to be characterised for the purposes of pharmaceutical biocatalysis. BC61-TAm is an (R)-selective enzyme, cloned from an extremely halophilic archaeon, isolated from a Triassic period salt mine. Produced using a Haloferax volcanii-based expression model, the resulting protein displays a classic halophilic activity profile, as well as thermotolerance (optimum 50 °C) and organic solvent tolerance. Molecular modelling predicts the putative active site residues of haloarchaeal TAms, with molecular dynamics simulations providing insights on the basis of BC61-TAm's organic solvent tolerance. These results represent an exciting advance in the study of transaminases from extremophiles, providing a possible scaffold for future discovery of biocatalytic enzymes with robust properties.


Assuntos
Archaea/enzimologia , Proteínas Arqueais/metabolismo , Mineração , Cloreto de Sódio , Transaminases/metabolismo , Aminas/metabolismo , Archaea/genética , Proteínas Arqueais/genética , Biocatálise , Haloferax volcanii/enzimologia , Haloferax volcanii/genética , Simulação de Dinâmica Molecular , Solventes/metabolismo , Especificidade por Substrato , Termotolerância , Transaminases/genética
2.
Appl Microbiol Biotechnol ; 103(9): 3807-3817, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30877354

RESUMO

Enzyme-mediated synthesis of pharmaceutical compounds is a 'green' alternative to traditional synthetic chemistry, and microbial engineering opens up the possibility of using whole cells as mini-factories. Whole-cell biocatalysis reduces cost by eliminating expensive enzyme purification and cofactor addition steps, as well as resulting in increased enzyme stability. Haloferax volcanii is a model halophilic archaeon encoding highly salt and organic solvent tolerant enzymes such as alcohol dehydrogenase (HvADH2), which catalyses the reduction of aldehydes and ketone in the presence of NADPH/NADH cofactor. A H. volcanii strain for constitutive HvADH2 expression was generated using a strong synthetic promoter (p.syn). The strain was immobilised in calcium alginate beads and repeatedly used as a whole-cell biocatalyst. The reduction of acetophenone, used as test substrate, was very successful and high yields were detected from immobilised whole cells over repeated biotransformation cycles. The immobilised H. volcanii retained stability and high product yields after 1 month of storage at room temperature. This newly developed system offers halophilic enzyme expression in its native environment, high product yield, stability and reusability without the addition of any expensive NADPH/NADH cofactor. This is the first report of whole cell-mediated biocatalysis by the halophilic archaeon H. volcanii.


Assuntos
Álcool Desidrogenase/química , Proteínas Arqueais/química , Haloferax volcanii/metabolismo , Sais/metabolismo , Acetofenonas/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Aldeídos/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Biocatálise , Células Imobilizadas/química , Células Imobilizadas/enzimologia , Células Imobilizadas/metabolismo , Estabilidade Enzimática , Expressão Gênica , Haloferax volcanii/química , Haloferax volcanii/enzimologia , Cetonas/metabolismo , NADP/metabolismo
3.
Nucleic Acids Res ; 46(17): 9027-9043, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30102394

RESUMO

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN's function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3'-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3'-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.


Assuntos
Proteínas Arqueais/química , Reparo do DNA , DNA de Cadeia Simples/química , Exonucleases/química , Pyrococcus furiosus/enzimologia , RNA Arqueal/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Cátions Bivalentes , Clonagem Molecular , Cristalografia por Raios X , Quebras de DNA de Cadeia Simples , Dano ao DNA , Replicação do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Expressão Gênica , Haloferax volcanii/química , Haloferax volcanii/efeitos dos fármacos , Haloferax volcanii/enzimologia , Haloferax volcanii/genética , Cinética , Manganês/química , Manganês/metabolismo , Metanossulfonato de Metila/farmacologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pyrococcus furiosus/química , Pyrococcus furiosus/efeitos dos fármacos , Pyrococcus furiosus/genética , RNA Arqueal/genética , RNA Arqueal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
4.
Nucleic Acids Res ; 46(16): 8483-8499, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30010922

RESUMO

Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms.


Assuntos
Proteínas Arqueais/fisiologia , Proteínas de Bactérias/fisiologia , Haloferax volcanii/enzimologia , Processamento Pós-Transcricional do RNA , tRNA Metiltransferases/fisiologia , Proteínas de Bactérias/genética , Cristalografia por Raios X , Conjuntos de Dados como Assunto , Ativação Enzimática , Células Eucarióticas/enzimologia , Evolução Molecular , Holoenzimas/fisiologia , Imunoprecipitação , Espectrometria de Massas , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Proteômica , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade da Espécie , tRNA Metiltransferases/deficiência , tRNA Metiltransferases/genética
5.
J Bacteriol ; 200(17)2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29914986

RESUMO

DeoR-type helix-turn-helix (HTH) domain proteins are transcriptional regulators of sugar and nucleoside metabolism in diverse bacteria and also occur in select archaea. In the model archaeon Haloferax volcanii, previous work implicated GlpR, a DeoR-type transcriptional regulator, in the transcriptional repression of glpR and the gene encoding the fructose-specific phosphofructokinase (pfkB) during growth on glycerol. However, the global regulon governed by GlpR remained unclear. Here, we compared transcriptomes of wild-type and ΔglpR mutant strains grown on glycerol and glucose to detect significant transcript level differences for nearly 50 new genes regulated by GlpR. By coupling computational prediction of GlpR binding sequences with in vivo and in vitro DNA binding experiments, we determined that GlpR directly controls genes encoding enzymes involved in fructose degradation, including fructose bisphosphate aldolase, a central control point in glycolysis. GlpR also directly controls other transcription factors. In contrast, other metabolic pathways appear to be under the indirect influence of GlpR. In vitro experiments demonstrated that GlpR purifies to function as a tetramer that binds the effector molecule fructose-1-phosphate (F1P). These results suggest that H. volcanii GlpR functions as a direct negative regulator of fructose degradation during growth on carbon sources other than fructose, such as glucose and glycerol, and that GlpR bears striking functional similarity to bacterial DeoR-type regulators.IMPORTANCE Many archaea are extremophiles, able to thrive in habitats of extreme salinity, pH and temperature. These biological properties are ideal for applications in biotechnology. However, limited knowledge of archaeal metabolism is a bottleneck that prevents the broad use of archaea as microbial factories for industrial products. Here, we characterize how sugar uptake and use are regulated in a species that lives in high salinity. We demonstrate that a key sugar regulatory protein in this archaeal species functions using molecular mechanisms conserved with distantly related bacterial species.


Assuntos
Proteínas Arqueais/genética , Frutose/metabolismo , Regulação da Expressão Gênica em Archaea , Haloferax volcanii/genética , Proteínas Repressoras/genética , Proteínas Arqueais/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucose/metabolismo , Glicerol/metabolismo , Haloferax volcanii/enzimologia , Redes e Vias Metabólicas , Mutação , Regulon , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética
6.
FEBS Lett ; 592(9): 1524-1534, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29572819

RESUMO

The halophilic archaeon Haloferax volcanii degrades glucose via the semiphosphorylative Entner-Doudoroff pathway and can also grow on gluconeogenic substrates. Here, the enzymes catalysing the conversion of glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate were analysed. The genome contains the genes gapI and gapII encoding two putative GAP dehydrogenases, and pgk encoding phosphoglycerate kinase (PGK). We show that gapI is functionally involved in sugar catabolism, whereas gapII is involved in gluconeogenesis. For pgk, an amphibolic function is indicated. This is the first report of the functional involvement of a phosphorylating glyceraldehyde-3-phosphate dehydrogenase and PGK in sugar catabolism in archaea. Phylogenetic analyses indicate that the catabolic gapI from H. volcanii is acquired from bacteria via lateral genetransfer, whereas the anabolic gapII as well as pgk are of archaeal origin.


Assuntos
Gluconeogênese , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicólise , Haloferax volcanii/metabolismo , Técnicas de Inativação de Genes , Gliceraldeído-3-Fosfato Desidrogenases/deficiência , Gliceraldeído-3-Fosfato Desidrogenases/genética , Haloferax volcanii/enzimologia
7.
J Proteome Res ; 17(3): 1158-1171, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29411617

RESUMO

The membrane protease LonB is an essential protein in the archaeon Haloferax volcanii and globally impacts its physiology. However, natural substrates of the archaeal Lon protease have not been identified. The whole proteome turnover was examined in a H. volcanii LonB mutant under reduced and physiological protease levels. LC-MS/MS combined with stable isotope labeling was applied for the identification/quantitation of membrane and cytoplasm proteins. Differential synthesis and degradation rates were evidenced for 414 proteins in response to Lon expression. A total of 58 proteins involved in diverse cellular processes showed a degradation pattern (none/very little degradation in the absence of Lon and increased degradation in the presence of Lon) consistent with a LonB substrate, which was further substantiated for several of these candidates by pull-down assays. The most notable was phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthesis. The rapid degradation of PSY upon LonB induction in addition to the remarkable stabilization of this protein and hyperpigmentation phenotype in the Lon mutant strongly suggest that PSY is a LonB substrate. This work identifies for the first time candidate targets of the archaeal Lon protease and establishes proteolysis by Lon as a novel post-translational regulatory mechanism of carotenogenesis.


Assuntos
Proteínas Arqueais/metabolismo , Carotenoides/biossíntese , Regulação da Expressão Gênica em Archaea , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Haloferax volcanii/enzimologia , Protease La/metabolismo , Proteoma/metabolismo , Proteínas Arqueais/genética , Cromatografia Líquida , Ontologia Genética , Geranil-Geranildifosfato Geranil-Geraniltransferase/genética , Haloferax volcanii/genética , Marcação por Isótopo/métodos , Anotação de Sequência Molecular , Mutação , Protease La/genética , Biossíntese de Proteínas , Proteólise , Proteoma/genética , Especificidade por Substrato , Espectrometria de Massas em Tandem
8.
Nucleic Acids Res ; 46(3): 1441-1456, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29237037

RESUMO

Eukaryotic ribosome biogenesis is a complex dynamic process which requires the action of numerous ribosome assembly factors. Among them, the eukaryotic Rio protein family members (Rio1, Rio2 and Rio3) belong to an ancient conserved atypical protein kinase/ ATPase family required for the maturation of the small ribosomal subunit (SSU). Recent structure-function analyses suggested an ATPase-dependent role of the Rio proteins to regulate their dynamic association with the nascent pre-SSU. However, the evolutionary origin of this feature and the detailed molecular mechanism that allows controlled activation of the catalytic activity remained to be determined. In this work we provide functional evidence showing a conserved role of the archaeal Rio proteins for the synthesis of the SSU in archaea. Moreover, we unravel a conserved RNA-dependent regulation of the Rio ATPases, which in the case of Rio2 involves, at least, helix 30 of the SSU rRNA and the P-loop lysine within the shared RIO domain. Together, our study suggests a ribosomal RNA-mediated regulatory mechanism enabling the appropriate stimulation of Rio2 catalytic activity and subsequent release of Rio2 from the nascent pre-40S particle. Based on our findings we propose a unified release mechanism for the Rio proteins.


Assuntos
Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Proteínas Arqueais/genética , Haloferax volcanii/enzimologia , Proteínas Serina-Treonina Quinases/genética , RNA Ribossômico 18S/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Evolução Molecular , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Haloferax volcanii/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , RNA Ribossômico 18S/química , RNA Ribossômico 18S/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Eur Biophys J ; 47(3): 225-236, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28875401

RESUMO

Laccase (benzenediol: oxygen oxidoreductases, EC1.10.3.2) is a multi-copper oxidase capable of oxidizing a variety of phenolic and other aromatic organic compounds. The catalytic power of laccase makes it an attractive candidate for potential applications in many areas of industry including biodegradation of organic pollutants and synthesis of novel drugs. Most laccases are vulnerable to high salt and have limited applications. However, some laccases are not only tolerant to but also activated by certain concentrations of salt and thus have great application potential. The mechanisms of salt-induced activity enhancement of laccases are unclear as yet. In this study, we used dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, intrinsic fluorescence emission, circular dichroism, ultraviolet-visible light absorption, and an enzymatic assay to investigate the potential correlation between the structure and activity of the marine-derived laccase, Lac15, whose activity is promoted by low concentrations of NaCl. The results showed that low concentrations of NaCl exert little influence on the protein structure, which was partially folded in the absence of the salt; moreover, the partially folded rather than the fully folded state seemed to be favorable for enzyme activity, and this partially folded state was distinctive from the so-called 'molten globule' occasionally observed in active enzymes. More data indicated that salt might promote laccase activity through mechanisms involving perturbation of specific local sites rather than a change in global structure. Potential binding sites for chloride ions and their roles in enzyme activity promotion are proposed.


Assuntos
Cloretos/farmacologia , Haloferax volcanii/enzimologia , Lacase/metabolismo , Cobre/metabolismo , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Lacase/química , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Especificidade por Substrato
10.
PLoS One ; 12(12): e0189913, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29284023

RESUMO

Halophilic euryarchaea lack many of the genes necessary for the protoporphyrin-dependent heme biosynthesis pathway previously identified in animals and plants. Bioinformatic analysis suggested the presence of two heme biosynthetic processes, an Fe-coproporphyrinogen III (coproheme) decarboxylase (ChdC) pathway and an alternative heme biosynthesis (Ahb) pathway, in Haloferax volcanii. PitA is specific to the halophilic archaea and has a unique molecular structure in which the ChdC domain is joined to the antibiotics biosynthesis monooxygenase (ABM)-like domain by a histidine-rich linker sequence. The pitA gene deletion variant of H. volcanii showed a phenotype with a significant reduction of aerobic growth. Addition of a protoheme complemented the phenotype, supporting the assumption that PitA participates in the aerobic heme biosynthesis. Deletion of the ahbD gene caused a significant reduction of only anaerobic growth by denitrification or dimethylsulfoxide (DMSO) respiration, and the growth was also complemented by addition of a protoheme. The experimental results suggest that the two heme biosynthesis pathways are utilized selectively under aerobic and anaerobic conditions in H. volcanii. The molecular structure and physiological function of PitA are also discussed on the basis of the limited proteolysis and sequence analysis.


Assuntos
Proteínas Arqueais/metabolismo , Haloferax volcanii/crescimento & desenvolvimento , Heme/metabolismo , Deleção de Genes , Regulação da Expressão Gênica em Archaea , Haloferax volcanii/enzimologia , Haloferax volcanii/genética
11.
PLoS One ; 12(11): e0187482, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29190711

RESUMO

An alcohol dehydrogenase from the halophilic archaeon Haloferax volcanii (HvADH2) has been engineered by rational design to broaden its substrate scope towards the conversion of a range of aromatic substrates, including flurbiprofenol, that is an intermediate of the non-steroidal anti-inflammatory drug, flurbiprofen. Wild-type HvADH2 showed minimal activity with flurbiprofenol (11.1 mU/mg). A homology model of HvADH2 was built and docking experiments with this substrate revealed that the biphenyl rings of flurbiprofenol formed strong interactions with residues F85 and F108, preventing its optimal binding in the active site. Mutations at position 85 however did not increase activity. Site directed mutagenesis at position F108 allowed the identification of three variants showing a significant (up to 2.3-fold) enhancement of activity towards flurbiprofenol, when compared to wild-type HvADH2. Interestingly, F108G variant did not show the classic inhibition in the presence of (R)-enantiomer when tested with rac-1-phenylethanol, underling its potential in racemic resolution of secondary alcohols.


Assuntos
Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/química , Álcool Desidrogenase/genética , Sequência de Aminoácidos , Simulação por Computador , Haloferax volcanii/enzimologia , Ligações de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
12.
Org Biomol Chem ; 15(43): 9169-9175, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-29067382

RESUMO

Enzymatic synthesis of enantiopure aromatic secondary alcohols (including substituted, hetero-aromatic and bicyclic structures) was carried out using halophilic alcohol dehydrogenase ADH2 from Haloferax volcanii (HvADH2). This enzyme showed an unprecedented substrate scope and absolute enatioselectivity. The cofactor NADPH was used catalytically and regenerated in situ by the biocatalyst, in the presence of 5% ethanol. The efficiency of HvADH2 for the conversion of aromatic ketones was markedly influenced by the steric and electronic factors as well as the solubility of ketones in the reaction medium. Furthermore, carbonyl stretching band frequencies ν (C[double bond, length as m-dash]O) have been measured for different ketones to understand the effect of electron withdrawing or donating properties of the ketone substituents on the reaction rate catalyzed by HvADH2. Good correlation was observed between ν (C[double bond, length as m-dash]O) of methyl aryl-ketones and the reaction rate catalyzed by HvADH2. The enzyme catalyzed the reductions of ketone substrates on the preparative scale, demonstrating that HvADH2 would be a valuable biocatalyst for the preparation of chiral aromatic alcohols of pharmaceutical interest.


Assuntos
Álcool Desidrogenase/metabolismo , Álcoois/química , Álcoois/síntese química , Biocatálise , Haloferax volcanii/enzimologia , Técnicas de Química Sintética
13.
FEMS Microbiol Lett ; 364(13)2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28854683

RESUMO

Haloferax volcanii degrades the pentoses D-xylose and L-arabinose via an oxidative pathway to α-ketoglutarate as an intermediate. The initial dehydrogenases of the pathway, D-xylose dehydrogenase (XDH) and L-arabinose dehydrogenase (L-AraDH) catalyze the NADP+ dependent D-xylose and L-arabinose oxidation. It is shown here that the pentoses are oxidized to the corresponding lactones, D-xylono-γ-lactone and L-arabino-γ-lactone, rather than to the respective sugar acids. A putative lactonase gene, xacC, located in genomic vicinity of XDH and L-AraDH, was found to be transcriptionally upregulated by both D-xylose and L-arabinose mediated by the pentose-specific regulator XacR. The recombinant lactonase catalyzed the hydrolysis of D-xylono-γ-lactone and L-arabino-γ-lactone. This is the first report of a functional lactonase involved in sugar catabolism in the domain of archaea.


Assuntos
Arabinose/metabolismo , Esterases/metabolismo , Haloferax volcanii/enzimologia , Xilose/metabolismo , Acil-Butirolactonas/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Desidrogenases de Carboidrato/genética , Desidrogenases de Carboidrato/metabolismo , Esterases/genética , Hidrólise , Ácidos Cetoglutáricos/metabolismo , Mutação , Oxirredução , RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulação para Cima
14.
DNA Repair (Amst) ; 55: 7-16, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28501701

RESUMO

Homologous recombination plays a central role in the repair of double-strand DNA breaks, the restart of stalled replication forks and the generation of genetic diversity. Regulation of recombination is essential since defects can lead to genome instability and chromosomal rearrangements. Strand exchange is a key step of recombination - it is catalysed by RecA in bacteria, Rad51/Dmc1 in eukaryotes and RadA in archaea. RadB, a paralogue of RadA, is present in many archaeal species. RadB has previously been proposed to function as a recombination mediator, assisting in RadA-mediated strand exchange. In this study, we use the archaeon Haloferax volcanii to provide evidence to support this hypothesis. We show that RadB is required for efficient recombination and survival following treatment with DNA-damaging agents, and we identify two point mutations in radA that suppress the ΔradB phenotype. Analysis of these point mutations leads us to propose that the role of RadB is to act as a recombination mediator, which it does by inducing a conformational change in RadA and thereby promoting its polymerisation on DNA.


Assuntos
Proteínas Arqueais/metabolismo , Quebras de DNA de Cadeia Dupla , Haloferax volcanii/enzimologia , Recombinases Rec A/metabolismo , Reparo de DNA por Recombinação , Sequência de Aminoácidos , Proteínas Arqueais/química , DNA Arqueal/metabolismo , Haloferax volcanii/genética , Recombinases Rec A/química , Alinhamento de Sequência
15.
FEBS J ; 283(19): 3563-3566, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27542853

RESUMO

The Small Archaeal Modifier Proteins (SAMPs) from Haloferax volcanii belong to the group of ubiquitin like proteins (Ubls) that act both as protein modifiers and sulfur carriers. The E1-like enzyme UbaA is essential for SAMP activation and therefore required for both sampylation and sulfur transfer. Here, we provide a commentary on the thorough characterization of UbaA by J. Maupin-Furlow and colleagues.


Assuntos
Haloferax volcanii/enzimologia , Enxofre , Proteínas Arqueais , Ubiquitinas
16.
RNA ; 22(10): 1604-19, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27539785

RESUMO

In Eukarya and Archaea, in addition to protein-only pseudouridine (Ψ) synthases, complexes containing one guide RNA and four proteins can also produce Ψ. Cbf5 protein is the Ψ synthase in the complex. Previously, we showed that Ψ's at positions 1940, 1942, and 2605 of Haloferax volcanii 23S rRNA are absent in a cbf5-deleted strain, and a plasmid-borne copy of cbf5 can rescue the synthesis of these Ψ's. Based on published reports of the structure of archaeal Cbf5 complexed with other proteins and RNAs, we identified several potential residues and structures in H. volcanii Cbf5, which were expected to play important roles in pseudouridylation. We mutated these structures and determined their effects on Ψ production at the three rRNA positions under in vivo conditions. Mutations of several residues in the catalytic domain and certain residues in the thumb loop either abolished Ψ's or produced partial modification; the latter indicates a slower rate of Ψ formation. The universal catalytic aspartate of Ψ synthases could be replaced by glutamate in Cbf5. A conserved histidine, which is common to Cbf5 and TruB is not needed, but another conserved histidine of Cbf5 is required for the in vivo RNA-guided Ψ formation. We also identified a previously unreported novelty in the pseudouridylation activity of Cbf5 where a single stem-loop of a guide H/ACA RNA is used to produce two closely placed Ψ's and mutations of certain residues of Cbf5 abolished one of these two Ψ's. In summary, this first in vivo study identifies several structures of an archaeal Cbf5 protein that are important for its RNA-guided pseudouridylation activity.


Assuntos
Proteínas Arqueais/química , Haloferax volcanii/enzimologia , Transferases Intramoleculares/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Domínio Catalítico , Sequência Conservada , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Mutação , RNA Ribossômico/metabolismo , Relação Estrutura-Atividade
17.
FEBS J ; 283(19): 3567-3586, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27459543

RESUMO

Here we provide the first detailed biochemical study of a noncanonical E1-like enzyme with broad specificity for cognate ubiquitin-like (Ubl) proteins that mediates Ubl protein modification and sulfur mobilization to form molybdopterin and thiolated tRNA. Isothermal titration calorimetry and in vivo analyses proved useful in discovering that environmental conditions, ATP binding, and Ubl type controlled the mechanism of association of the Ubl protein with its cognate E1-like enzyme (SAMP and UbaA of the archaeon Haloferax volcanii, respectively). Further analysis revealed that ATP hydrolysis triggered the formation of thioester and peptide bonds within the Ubl:E1-like complex. Importantly, the thioester was an apparent precursor to Ubl protein modification but not sulfur mobilization. Comparative modeling to MoeB/ThiF guided the discovery of key residues within the adenylation domain of UbaA that were needed to bind ATP as well as residues that were specifically needed to catalyze the downstream reactions of sulfur mobilization and/or Ubl protein modification. UbaA was also found to be Ubl-automodified at lysine residues required for early (ATP binding) and late (sulfur mobilization) stages of enzyme activity revealing multiple layers of autoregulation. Cysteine residues, distinct from the canonical E1 'active site' cysteine, were found important in UbaA function supporting a model that this noncanonical E1 is structurally flexible in its active site to allow Ubl~adenylate, Ubl~E1-like thioester and cysteine persulfide(s) intermediates to form.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Enxofre/metabolismo , Enzimas Ativadoras de Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/metabolismo , Trifosfato de Adenosina/metabolismo , Cisteína/fisiologia , Haloferax volcanii/enzimologia , Ligantes , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Compostos de Sulfidrila/metabolismo , Termodinâmica , Ubiquitinação
18.
J Bacteriol ; 198(16): 2251-62, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27297879

RESUMO

UNLABELLED: The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcanii IMPORTANCE: The work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii The functional involvement of the three enzymes was proven by analyses of the corresponding knockout mutants. These results provide evidence for the in vivo operation of the semiphosphorylative Entner-Doudoroff pathway in haloarchaea and thus expand our understanding of the unusual sugar degradation pathways in the domain Archaea.


Assuntos
Aldeído Liases/metabolismo , Proteínas Arqueais/metabolismo , Regulação da Expressão Gênica em Archaea/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucose 1-Desidrogenase/metabolismo , Haloferax volcanii/enzimologia , Hidroliases/metabolismo , Aldeído Liases/genética , Sequência de Aminoácidos , Proteínas Arqueais/genética , Deleção de Genes , Glucose 1-Desidrogenase/genética , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Hidroliases/genética , Filogenia
19.
Appl Environ Microbiol ; 82(2): 538-48, 2016 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-26546423

RESUMO

Soluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PPi) to orthophosphate (Pi) are commonly used to accelerate and detect biosynthetic reactions that generate PPi as a by-product. Current PPAs are inactivated by high salt concentrations and organic solvents, which limits the extent of their use. Here we report a class A type PPA of the haloarchaeon Haloferax volcanii (HvPPA) that is thermostable and displays robust PPi-hydrolyzing activity under conditions of 25% (vol/vol) organic solvent and salt concentrations from 25 mM to 3 M. HvPPA was purified to homogeneity as a homohexamer by a rapid two-step method and was found to display non-Michaelis-Menten kinetics with a Vmax of 465 U · mg(-1) for PPi hydrolysis (optimal at 42°C and pH 8.5) and Hill coefficients that indicated cooperative binding to PPi and Mg(2+). Similarly to other class A type PPAs, HvPPA was inhibited by sodium fluoride; however, hierarchical clustering and three-dimensional (3D) homology modeling revealed HvPPA to be distinct in structure from characterized PPAs. In particular, HvPPA was highly negative in surface charge, which explained its extreme resistance to organic solvents. To demonstrate that HvPPA could drive thermodynamically unfavorable reactions to completion under conditions of reduced water activity, a novel coupled assay was developed; HvPPA hydrolyzed the PPi by-product generated in 2 M NaCl by UbaA (a "salt-loving" noncanonical E1 enzyme that adenylates ubiquitin-like proteins in the presence of ATP). Overall, we demonstrate HvPPA to be useful for hydrolyzing PPi under conditions of reduced water activity that are a hurdle to current PPA-based technologies.


Assuntos
Proteínas Arqueais/metabolismo , Haloferax volcanii/enzimologia , Pirofosfatase Inorgânica/metabolismo , Cloreto de Sódio/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Estabilidade Enzimática , Haloferax volcanii/química , Haloferax volcanii/classificação , Haloferax volcanii/genética , Temperatura Alta , Pirofosfatase Inorgânica/química , Pirofosfatase Inorgânica/genética , Cinética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Cloreto de Sódio/análise , Solventes/química , Solventes/metabolismo , Especificidade por Substrato
20.
Appl Microbiol Biotechnol ; 100(3): 1183-1195, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26428236

RESUMO

The success of biotechnological processes is based on the availability of efficient and highly specific biocatalysts, which can satisfy industrial demands. Extreme and remote environments like the deep brine pools of the Red Sea represent highly interesting habitats for the discovery of novel halophilic and thermophilic enzymes. Haloferax volcanii constitutes a suitable expression system for halophilic enzymes obtained from such brine pools. We developed a batch process for the cultivation of H. volcanii H1895 in controlled stirred-tank bioreactors utilising knockouts of components of the flagella assembly system. The standard medium Hv-YPC was supplemented to reach a higher cell density. Without protein expression, cell dry weight reaches 10 g L(-1). Two halophilic alcohol dehydrogenases were expressed under the control of the tryptophanase promoter p.tna with 16.8 and 3.2 mg gCDW (-1), respectively, at a maximum cell dry weight of 6.5 g L(-1). Protein expression was induced by the addition of L-tryptophan. Investigation of various expression strategies leads to an optimised two-step induction protocol introducing 6 mM L-tryptophan at an OD650 of 0.4 followed by incubation for 16 h and a second induction step with 3 mM L-tryptophan followed by a final incubation time of 4 h. Compared with the uncontrolled shaker-flask cultivations used until date, dry cell mass concentrations were improved by a factor of more than 5 and cell-specific enzyme activities showed an up to 28-fold increased yield of the heterologous proteins.


Assuntos
Álcool Desidrogenase/biossíntese , Proteínas Arqueais/biossíntese , Reatores Biológicos/microbiologia , Haloferax volcanii/enzimologia , Microbiologia Industrial/métodos , Álcool Desidrogenase/química , Álcool Desidrogenase/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Estabilidade Enzimática , Haloferax volcanii/química , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Microbiologia Industrial/instrumentação , Triptofano/metabolismo
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