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1.
Appl Microbiol Biotechnol ; 104(4): 1371-1382, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31863144

RESUMO

Haloferax volcanii is an obligate halophilic archaeon with its origin in the Dead Sea. Simple laboratory culture conditions and a wide range of genetic tools have made it a model organism for studying haloarchaeal cell biology. Halophilic enzymes of potential interest to biotechnology have opened up the application of this organism in biocatalysis, bioremediation, nanobiotechnology, bioplastics and the biofuel industry. Functionally active halophilic proteins can be easily expressed in a halophilic environment, and an extensive genetic toolkit with options for regulated protein overexpression has allowed the purification of biotechnologically important enzymes from different halophiles in H. volcanii. However, corrosion mediated damage caused to stainless-steel bioreactors by high salt concentrations and a tendency to form biofilms when cultured in high volume are some of the challenges of applying H. volcanii in biotechnology. The ability to employ expressed active proteins in immobilized cells within a porous biocompatible matrix offers new avenues for exploiting H. volcanii in biotechnology. This review critically evaluates the various application potentials, challenges and toolkits available for using this extreme halophilic organism in biotechnology.


Assuntos
Haloferax volcanii/enzimologia , Haloferax volcanii/genética , Microbiologia Industrial/tendências , Biocatálise , Biofilmes , Reatores Biológicos/microbiologia , Células Imobilizadas , Proteômica
2.
IUBMB Life ; 71(8): 1109-1116, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31283101

RESUMO

The mature 5'-ends of tRNAs are generated by RNase P in all domains of life. The ancient form of the enzyme is a ribonucleoprotein consisting of a catalytic RNA and one or more protein subunits. However, in the hyperthermophilic bacterium Aquifex aeolicus and close relatives, RNase P is a protein-only enzyme consisting of a single type of polypeptide (Aq_880, ~23 kDa). In many archaea, homologs of Aq_880 were identified (termed HARPs for Homologs of Aquifex RNase P) in addition to the RNA-based RNase P, raising the question about the functions of HARP and the classical RNase P in these archaea. Here we investigated HARPs from two euryarchaeotes, Haloferax volcanii and Methanosarcina mazei. Archaeal strains with HARP gene knockouts showed no growth phenotypes under standard conditions, temperature and salt stress (H. volcanii) or nitrogen deficiency (M. mazei). Recombinant H. volcanii and M. mazei HARPs were basically able to catalyse specific tRNA 5'-end maturation in vitro. Furthermore, M. mazei HARP was able to rescue growth of an Escherichia coli RNase P depletion strain with comparable efficiency as Aq_880, while H. volcanii HARP was unable to do so. In conclusion, both archaeal HARPs showed the capacity (in at least one functional assay) to act as RNases P. However, the ease to obtain knockouts of the singular HARP genes and the lack of growth phenotypes upon HARP gene deletion contrasts with the findings that the canonical RNase P RNA gene cannot be deleted in H. volcanii, and a knockdown of RNase P RNA in H. volcanii results in severe tRNA processing defects. We conclude that archaeal HARPs do not make a major contribution to global tRNA 5'-end maturation in archaea, but may well exert a specialised, yet unknown function in (t)RNA metabolism. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1109-1116, 2019.


Assuntos
Bactérias/enzimologia , Haloferax volcanii/enzimologia , Methanosarcina/enzimologia , Ribonuclease P/metabolismo , Catálise , Dicroísmo Circular , Escherichia coli/metabolismo , Deleção de Genes , Teste de Complementação Genética , Conformação de Ácido Nucleico , Fenótipo , Plasmídeos/genética , RNA de Transferência/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Temperatura , Thermus thermophilus/enzimologia
3.
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
4.
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
5.
Microbiologyopen ; 8(9): e00829, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30884174

RESUMO

The role of cyclic nucleotides as second messengers for intracellular signal transduction has been well described in bacteria. One recently discovered bacterial second messenger is cyclic di-adenylate monophosphate (c-di-AMP), which has been demonstrated to be essential in bacteria. Compared to bacteria, significantly less is known about second messengers in archaea. This study presents the first evidence of in vivo presence of c-di-AMP in an archaeon. The model organism Haloferax volcanii was demonstrated to produce c-di-AMP. Its genome encodes one diadenylate cyclase (DacZ) which was shown to produce c-di-AMP in vitro. Similar to bacteria, the dacZ gene is essential and homologous overexpression of DacZ leads to cell death, suggesting the need for tight regulation of c-di-AMP levels. Such tight regulation often indicates the control of important regulatory processes. A central target of c-di-AMP signaling in bacteria is cellular osmohomeostasis. The results presented here suggest a comparable function in H. volcanii. A strain with decreased c-di-AMP levels exhibited an increased cell area in hypo-salt medium, implying impaired osmoregulation. In summary, this study expands the field of research on c-di-AMP and its physiological function to archaea and indicates that osmoregulation is likely to be a common function of c-di-AMP in bacteria and archaea.


Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Haloferax volcanii/metabolismo , Adenilil Ciclases/genética , Regulação da Expressão Gênica em Archaea , Genes Essenciais , Genoma Bacteriano , Haloferax volcanii/enzimologia , Haloferax volcanii/genética , Osmorregulação , Transdução de Sinais
6.
Mol Microbiol ; 111(4): 1093-1108, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30707467

RESUMO

The halophilic archaeon Haloferax volcanii utilizes l-rhamnose as a sole carbon and energy source. It is shown that l-rhamnose is taken up by an ABC transporter and is oxidatively degraded to pyruvate and l-lactate via the diketo-hydrolase pathway. The genes involved in l-rhamnose uptake and degradation form a l-rhamnose catabolism (rhc) gene cluster. The rhc cluster also contains a gene, rhcR, that encodes the transcriptional regulator RhcR which was characterized as an activator of all rhc genes. 2-keto-3-deoxy-l-rhamnonate, a metabolic intermediate of l-rhamnose degradation, was identified as inducer molecule of RhcR. The essential function of rhc genes for uptake and degradation of l-rhamnose was proven by the respective knockout mutants. Enzymes of the diketo-hydrolase pathway, including l-rhamnose dehydrogenase, l-rhamnonolactonase, l-rhamnonate dehydratase, 2-keto-3-deoxy-l-rhamnonate dehydrogenase and 2,4-diketo-3-deoxy-l-rhamnonate hydrolase, were characterized. Further, genes of the diketo-hydrolase pathway were also identified in the hyperthermophilic crenarchaeota Vulcanisaeta distributa and Sulfolobus solfataricus and selected enzymes were characterized, indicating the presence of the diketo-hydrolase pathway in these archaea. Together, this is the first comprehensive description of l-rhamnose catabolism in the domain of archaea.


Assuntos
Genes Arqueais , Haloferax volcanii/enzimologia , Haloferax volcanii/genética , Ramnose/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Desidrogenases de Carboidrato/metabolismo , Metabolismo dos Carboidratos , Família Multigênica , Oxirredutases/metabolismo , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismo
7.
Environ Microbiol ; 21(1): 286-298, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30370585

RESUMO

The model haloarchaeon, Haloferax volcanii possess an extremely high, and highly specific, basal caspase activity in exponentially growing cells that closely resembles caspase-4. This activity is specifically inhibited by the pan-caspase inhibitor, z-VAD-FMK, and has no cross-reactivity with other known protease families. Although it is one of the dominant cellular proteolytic activities in exponentially growing H. volcanii cells, the interactive cellular roles remain unknown and the protein(s) responsible for this activity remain elusive. Here, biochemical purification and in situ trapping with caspase targeted covalent inhibitors combined with genome-enabled proteomics, structural analysis, targeted gene knockouts and treatment with canavanine demonstrated a catalytic linkage between caspase activity and thermosomes, proteasomes and cdc48b, a cell division protein and proteasomal degradation facilitating ATPase, as part of an 'interactase' of stress-related protein complexes with an established link to the unfolded protein response (UPR). Our findings provide novel cellular and biochemical context for the observed caspase activity in Archaea and add new insight to understanding the role of this activity, implicating their possible role in the establishment of protein stress and ER associated degradation pathways in Eukarya.


Assuntos
Caspases/metabolismo , Haloferax volcanii/enzimologia , Proteostase/fisiologia , Adenosina Trifosfatases/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase/farmacologia , Ativação Enzimática/efeitos dos fármacos , Haloferax volcanii/efeitos dos fármacos , Haloferax volcanii/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteômica , Proteostase/efeitos dos fármacos
8.
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
9.
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
10.
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
11.
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
12.
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
13.
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
14.
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
15.
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
16.
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 , Ligação de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
17.
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
18.
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
19.
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
20.
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
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