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1.
Appl Environ Microbiol ; 84(9)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29475867

RESUMO

In many pro- and eukaryotes, a retinal-based proton pump equips the cell to drive ATP synthesis with (sun)light. Such pumps, therefore, have been proposed as a plug-in for cyanobacteria to artificially increase the efficiency of oxygenic photosynthesis. However, little information on the metabolism of retinal, their chromophore, is available for these organisms. We have studied the in vivo roles of five genes (sll1541, slr1648, slr0091, slr1192, and slr0574) potentially involved in retinal metabolism in Synechocystis sp. strain PCC 6803. With a gene deletion approach, we have shown that Synechocystis apo-carotenoid-15,15-oxygenase (SynACO), encoded by gene sll1541, is an indispensable enzyme for retinal synthesis in Synechocystis, presumably via asymmetric cleavage of ß-apo-carotenal. The second carotenoid oxygenase (SynDiox2), encoded by gene slr1648, competes with SynACO for substrate(s) but only measurably contributes to retinal biosynthesis in stationary phase via an as-yet-unknown mechanism. In vivo degradation of retinal may proceed through spontaneous chemical oxidation and via enzyme-catalyzed processes. Deletion of gene slr0574 (encoding CYP120A1), but not of slr0091 or of slr1192, causes an increase (relative to the level in wild-type Synechocystis) in the retinal content in both the linear and stationary growth phases. These results suggest that CYP120A1 does contribute to retinal degradation. Preliminary data obtained using 13C-labeled retinal suggest that conversion to retinol and retinoic acid and subsequent further oxidation also play a role. Deletion of sll1541 leads to deficiency in retinal synthesis and allows the in vivo reconstitution of far-red-absorbing holo-proteorhodopsin with exogenous retinal analogues, as demonstrated here for all-trans 3,4-dehydroretinal and 3-methylamino-16-nor-1,2,3,4-didehydroretinal.IMPORTANCE Retinal is formed by many cyanobacteria and has a critical role in most forms of life for processes such as photoreception, growth, and stress survival. However, the metabolic pathways in cyanobacteria for synthesis and degradation of retinal are poorly understood. In this paper we identify genes involved in its synthesis, characterize their role, and provide an initial characterization of the pathway of its degradation. This led to the identification of sll1541 (encoding SynACO) as the essential gene for retinal synthesis. Multiple pathways for retinal degradation presumably exist. These results have allowed us to construct a strain that expresses a light-dependent proton pump with an action spectrum extending beyond 700 nm. The availability of this strain will be important for further work aimed at increasing the overall efficiency of oxygenic photosynthesis.


Assuntos
Proteínas de Bactérias/genética , Sequência de Bases , Deleção de Sequência , Synechocystis/genética , Proteínas de Bactérias/biossíntese , Expressão Gênica , Rodopsinas Microbianas , Synechocystis/metabolismo
2.
ChemSusChem ; 10(10): 2267-2273, 2017 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-28425669

RESUMO

We introduce the concept of using site-specific sulfation of various lignins for increasing their aqueous solubility and thereby their processability. Using p-nitrophenylsulfate as a sulfate source and an aryl sulfotransferase enzyme as catalyst, lignins are easily sulfated at ambient conditions. We demonstrate the specific sulfation of phenolic hydroxyl groups on five different lignins: Indulin AT (Kraft softwood), Protobind 1000 (mixed wheat straw/Sarkanda grass soda) and three organosolv lignins. The reaction proceeds smoothly and the increase in solubility is visible to the naked eye. We then examine the reaction kinetics, and show that these are easily monitored qualitatively and quantitatively using UV/Vis spectroscopy. The UV/Vis results are validated with 31 P NMR spectroscopy of the lignin phenol groups after derivatization with phosphorylation reagent II. In general, the results are more significant with organosolv lignins, as Kraft and soda lignins are produced from aqueous lignocellulose extraction processes.


Assuntos
Arilsulfotransferase/química , Lignina/química , Espectroscopia de Ressonância Magnética , Espectrofotometria Ultravioleta , Água/química
3.
Epilepsia ; 57(5): 688-97, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26924447

RESUMO

OBJECTIVE: Inhibition of the mammalian target of rapamycin (mTOR) pathway has been suggested as a possible antiepileptogenic strategy in temporal lobe epilepsy (TLE). Here we aim to elucidate whether mTOR inhibition has antiepileptogenic and/or antiseizure effects using different treatment strategies in the electrogenic post-status epilepticus (SE) rat model. METHODS: Effects of mTOR inhibitor rapamycin were tested using the following three treatment protocols: (1) "stop-treatment"-post-SE treatment (6 mg/kg/day) was discontinued after 3 weeks; rats were monitored for 5 more weeks thereafter, (2) "pretreatment"-rapamycin (3 mg/kg/day) was applied during 3 days preceding SE; and (3) "chronic phase-treatment"-5 days rapamycin treatment (3 mg/kg/day) in the chronic phase. We also tested curcumin, an alternative mTOR inhibitor with antiinflammatory and antioxidant effects, using chronic phase treatment. Seizures were continuously monitored using video-electroencephalography (EEG) recordings; mossy fiber sprouting, cell death, and inflammation were studied using immunohistochemistry. Blood was withdrawn regularly to assess rapamycin and curcumin levels with high performance liquid chromatography (HPLC). RESULTS: Stop-treatment led to a strong reduction of seizures during the 3-week treatment and a gradual reappearance of seizures during the following 5 weeks. Three days pretreatment did not prevent seizure development, whereas 5-day rapamycin treatment in the chronic phase reduced seizure frequency. Washout of rapamycin was slow and associated with a gradual reappearance of seizures. Rapamycin treatment (both 3 and 6 mg/kg) led to body growth reduction. Curcumin treatment did not reduce seizure frequency or lead to a decrease in body weight. SIGNIFICANCE: The present study indicates that rapamycin cannot prevent epilepsy in the electrical stimulation post-SE rat model but has seizure-suppressing properties as long as rapamycin blood levels are sufficiently high. Oral curcumin treatment had no effect on chronic seizures, possibly because it did not reach the brain at adequate levels.


Assuntos
Anticonvulsivantes/uso terapêutico , Curcumina/uso terapêutico , Estimulação Elétrica/efeitos adversos , Sirolimo/uso terapêutico , Estado Epiléptico/tratamento farmacológico , Análise de Variância , Animais , Anticonvulsivantes/sangue , Peso Corporal/efeitos dos fármacos , Curcumina/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Eletroencefalografia , Hipocampo/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Sirolimo/sangue , Estado Epiléptico/sangue , Estado Epiléptico/etiologia , Fatores de Tempo , Resultado do Tratamento
4.
J Endod ; 39(8): 1035-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23880272

RESUMO

INTRODUCTION: Previous research showed an antimicrobial effect of vanadium chloroperoxidase (VCPO) on in vitro Enterococcus faecalis biofilms. The current study aimed to optimize the use of this enzyme at the root canal pH using a modified VCPO (mVCPO) that was adapted to function at a higher pH and to explore the biocompatibility of mVCPO. METHODS: The activity of the original and modified VCPO was assessed using the monochlorodimedone assay. For antimicrobial assessment, 48-hour biofilms of E. faecalis OS-16 were incubated 5 or 30 minutes with mVCPO, bromide, and hydrogen peroxide, and colony-forming units were determined. A metabolic activity assay was used to evaluate the cytotoxic effect of mVCPO on oral fibroblasts. RESULTS: Reaction products generated by mVCPO at a root canal pH of 7.7 significantly inactivated the biofilm after 5 minutes and even more after 30 minutes (Mann-Whitney U test, P < .05). The mVCPO reaction products showed less cytotoxic effects than control solutions and 0.5% sodium hypochlorite (Kruskal-Wallis test, P < .05). CONCLUSIONS: The incubation of mVCPO in the presence of its substrates with in vitro E. faecalis biofilms showed a significant antimicrobial effect at the root canal pH. Also, cytotoxicity tests showed preliminary biocompatibility. Therefore, an interappointment dressing containing mVCPO could aid in improving current endodontic treatment through continuous and local generation of antimicrobials.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Cloreto Peroxidase/farmacologia , Cavidade Pulpar/microbiologia , Enterococcus faecalis/efeitos dos fármacos , Antibacterianos/toxicidade , Materiais Biocompatíveis/farmacologia , Materiais Biocompatíveis/toxicidade , Brometos/farmacologia , Cloreto Peroxidase/toxicidade , Cicloexanonas , Cavidade Pulpar/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Teste de Materiais , Mucosa Bucal/citologia , Mucosa Bucal/efeitos dos fármacos , Oxidantes/farmacologia , Irrigantes do Canal Radicular/farmacologia , Irrigantes do Canal Radicular/toxicidade , Hipoclorito de Sódio/farmacologia , Fatores de Tempo
5.
ChemSusChem ; 5(12): 2348-53, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23150241

RESUMO

Herein, we report a new flow process with immobilized enzymes to synthesize complex chiral carbohydrate analogues from achiral inexpensive building blocks in a three-step cascade reaction. The first reactor contained immobilized acid phosphatase, which phosphorylated dihydroxyacetone to dihydroxyacetone phosphate using pyrophosphate as the phosphate donor. The second flow reactor contained fructose-1,6-diphosphate aldolase (RAMA, rabbit muscle aldolase) or rhamnulose-1-phosphate aldolase (RhuA from Thermotoga maritima) and acid phosphatase. The immobilized aldolases coupled the formed dihydroxyacetone phosphate to aldehydes, resulting in phosphorylated carbohydrates. A final reactor containing acid phosphatase that dephosphorylated the phosphorylated product yielded the final product. Different aldehydes were used to synthesize carbohydrates on a gram scale. To demonstrate the feasibility of the flow systems, we synthesized 0.6 g of the D-fagomine precursor. By using immobilized aldolase RhuA we were also able to obtain other stereoisomers of the D-fagomine precursor.


Assuntos
Fosfatase Ácida/química , Aldeído Liases/química , Carboidratos/síntese química , Enzimas Imobilizadas/química , Frutose-Bifosfato Aldolase/química , Compostos Organofosforados/síntese química , Aldeídos/química , Carboidratos/química , Fosfato de Di-Hidroxiacetona/química , Estrutura Molecular , Compostos Organofosforados/química , Estereoisomerismo
6.
Nat Nanotechnol ; 7(8): 530-5, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22751222

RESUMO

Marine biofouling--the colonization of small marine microorganisms on surfaces that are directly exposed to seawater, such as ships' hulls--is an expensive problem that is currently without an environmentally compatible solution. Biofouling leads to increased hydrodynamic drag, which, in turn, causes increased fuel consumption and greenhouse gas emissions. Tributyltin-free antifouling coatings and paints based on metal complexes or biocides have been shown to efficiently prevent marine biofouling. However, these materials can damage the environment through metal leaching (for example, of copper and zinc) and bacteria resistance. Here, we show that vanadium pentoxide nanowires act like naturally occurring vanadium haloperoxidases to prevent marine biofouling. In the presence of bromide ions and hydrogen peroxide, the nanowires catalyse the oxidation of bromide ions to hypobromous acid (HOBr). Singlet molecular oxygen ((1)O(2)) is formed and this exerts strong antibacterial activity, which prevents marine biofouling without being toxic to marine biota. Vanadium pentoxide nanowires have the potential to be an alternative approach to conventional anti-biofouling agents.


Assuntos
Incrustação Biológica , Nanopartículas , Nanofios , Compostos de Vanádio/química , Antibacterianos/química , Biofilmes/crescimento & desenvolvimento , Brometos/química , Humanos , Peróxido de Hidrogênio/química , Nanopartículas/química , Nanopartículas/microbiologia , Nanofios/química , Nanofios/microbiologia , Peroxidases/química , Água do Mar , Navios , Oxigênio Singlete/química
7.
Chemistry ; 18(21): 6604-9, 2012 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-22505143

RESUMO

Acid phosphatase, an enzyme that is able to catalyze the transfer of a phosphate group from cheap pyrophosphate to alcoholic substrates, was covalently immobilized on polymethacrylate beads with an epoxy linker (Immobeads-150 or Sepabeads EC-EP). After immobilization 70% of the activity was retained and the immobilized enzyme was stable for many months. With the immobilized enzyme we were able to produce and prepare D-glucose-6-phosphate, N-acetyl-D-glucosamine-6-phosphate, allyl phosphate, dihydroxyacetone phosphate, glycerol-1-phosphate, and inosine-5'-monophosphate from the corresponding primary alcohol on gram scale using either a fed-batch reactor or a continuous-flow packed-bed reactor.


Assuntos
Fosfatase Ácida/metabolismo , Compostos Organofosforados/síntese química , Acetilglucosamina/análogos & derivados , Acetilglucosamina/síntese química , Acetilglucosamina/química , Catálise , Glucose-6-Fosfato/síntese química , Glucose-6-Fosfato/química , Inosina Monofosfato/química , Compostos Organofosforados/química , Ácidos Polimetacrílicos/química
8.
J Endod ; 38(1): 72-4, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22152624

RESUMO

INTRODUCTION: The aim of this study was to explore the antimicrobial effect of vanadium chloroperoxidase (VCPO) reaction products on Enterococcus faecalis biofilms of 4 different strains. METHODS: Twenty-four-hour biofilms of E. faecalis strains V583, ER5/1, E2, and OS-16 were incubated in mixtures with VCPO, halide (either bromide or chloride), and hydrogen peroxide. The antibacterial efficacy was assessed by colony-forming unit counts. RESULTS: The VCPO reaction products had a similar efficacy in reducing the viability of the 4 strains of E. faecalis (94%; range, 87%-100%). Bromide as the halogen of choice was more effective on E. faecalis strains E2 and OS-16, as compared with chloride (Mann-Whitney U test; P < .05). Despite different quantities of produced biofilms by the 4 strains, VCPO treatment was similarly effective toward all strains (Kruskal-Wallis test; P < .05). CONCLUSIONS: VCPO treatment results in an antimicrobial effect toward in vitro E. faecalis biofilms and might provide an addition to current endodontic treatment, possibly as an antimicrobial dressing.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Cloreto Peroxidase/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Carga Bacteriana/efeitos dos fármacos , Técnicas Bacteriológicas , Brometos/farmacologia , Cloretos/farmacologia , Enterococcus faecalis/classificação , Humanos , Peróxido de Hidrogênio/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Hipoclorito de Sódio/farmacologia
9.
Chembiochem ; 10(13): 2230-5, 2009 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-19623595

RESUMO

To enhance the phosphorylating activity of the bacterial nonspecific acid phosphatase from Salmonella enterica ser. typhimurium LT2 towards dihydroxyacetone (DHA), a mutant library was generated from the native enzyme. Three different variants that showed enhanced activity were identified after one round of epPCR. The single mutant V78L was the most active and showed an increase in the maximal DHAP concentration to 25 % higher than that of the wild-type enzyme at pH 6.0. This variant is 17 times more active than the wild-type acid phosphatase from Salmonella enterica ser. typhimurium LT2 in the acid phosphatase/aldolase cascade reaction at pH 6.0 and is also six times more active than the phosphatase from Shigella flexneri that we previously used.


Assuntos
Fosfatase Ácida/metabolismo , Di-Hidroxiacetona/metabolismo , Evolução Molecular Direcionada , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Di-Hidroxiacetona/química , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Fosforilação , Conformação Proteica , Salmonella enterica/enzimologia
10.
Chem Biol ; 15(2): 189-99, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18291323

RESUMO

The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role in prokaryotic cell division and eukaryotic chromosome segregation, respectively. Selective inhibitors of the GTP-dependent polymerization of FtsZ could constitute a new class of antibiotics, while several inhibitors of tubulin are widely used in antiproliferative therapy. In this work, we set out to identify selective inhibitors of FtsZ based on the structure of its natural ligand, GTP. We found that GTP analogs with small hydrophobic substituents at C8 of the nucleobase efficiently inhibit FtsZ polymerization, whereas they have an opposite effect on the polymerization of tubulin. The inhibitory activity of the GTP analogs on FtsZ polymerization allowed us to crystallize FtsZ in complex with C8-morpholino-GTP, revealing the binding mode of a GTP derivative containing a nonmodified triphosphate chain.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas de Bactérias/química , Ligação Competitiva , Cristalografia por Raios X , Proteínas do Citoesqueleto/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Hidrólise , Polímeros/metabolismo , Tubulina (Proteína)/química , Moduladores de Tubulina/metabolismo
11.
Chembiochem ; 8(11): 1281-92, 2007 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-17600791

RESUMO

Cross-links between amino acid residues in close proximity can provide distance constraints for the validation of models of the 3D structure proteins. The mapping of cross-links by the identification of linked peptides in proteolytic digests is facilitated by cleavable cross-linkers that enable isolation of the cleavage products while preserving information about the linkage. We present an amine-specific cross-linker, bis(succinimidyl)-3-azidomethyl glutarate (BAMG), that fulfils these requirements. Two parallel reaction pathways are induced by tris(carboxyethyl)phosphine (TCEP) in cross-linked peptides from BAMG-treated cytochrome c. One pathway leads to cleavage of the cross-linked species, while in the other the azido group of BAMG is reduced to an amino group without cleavage. Cross-linked peptides and peptides modified by partially hydrolysed BAMG yield distinct sets of TCEP-induced reaction products. These can be isolated by reversed-phase diagonal chromatography and identified by mass spectrometry to reveal the identity of the parent compounds. The ease with which cross-link-derived reaction products can be isolated and identified indicates that the mapping of cross-links in complex biological assemblies and mixtures of protein complexes might become feasible in the near future.


Assuntos
Azidas/química , Reagentes para Ligações Cruzadas/química , Citocromos c/química , Lisina/química , Animais , Cromatografia Líquida de Alta Pressão , Cavalos , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/química , Fosfinas/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
12.
J Org Chem ; 71(16): 6244-7, 2006 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-16872211

RESUMO

A new enzymatic method for the generation of dihydroxyacetone phosphate (DHAP) using the acid phosphatase from Shigella flexneri (PhoN-Sf) and the cheap phosphate donor pyrophosphate (PPi) is described. The utility of this method was demonstrated in an aldolase-catalyzed condensation carried out in one pot in which DHAP was generated and coupled to propionaldehyde to give a yield of 53% of the isolated dephosphorylated end product.


Assuntos
Metabolismo dos Carboidratos , Carboidratos/síntese química , Fosfato de Di-Hidroxiacetona/química , Fosfato de Di-Hidroxiacetona/metabolismo , Fosfatos/química , Aldeídos/química , Carboidratos/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Shigella flexneri/enzimologia
13.
J Biol Chem ; 281(14): 9738-44, 2006 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-16455658

RESUMO

Directed evolution was performed on vanadium chloroperoxidase from the fungus Curvularia inaequalis to increase its brominating activity at a mildly alkaline pH for industrial and synthetic applications and to further understand its mechanism. After successful expression of the enzyme in Escherichia coli, two rounds of screening and selection, saturation mutagenesis of a "hot spot," and rational recombination, a triple mutant (P395D/L241V/T343A) was obtained that showed a 100-fold increase in activity at pH 8 (k(cat) = 100 s(-1)). The increased K(m) values for Br(-) (3.1 mm) and H(2)O(2) (16 microm) are smaller than those found for vanadium bromoperoxidases that are reasonably active at this pH. In addition the brominating activity at pH 5 was increased by a factor of 6 (k(cat) = 575 s(-1)), and the chlorinating activity at pH 5 was increased by a factor of 2 (k(cat) = 36 s(-1)), yielding the "best" vanadium haloperoxidase known thus far. The mutations are in the first and second coordination sphere of the vanadate cofactor, and the catalytic effects suggest that fine tuning of residues Lys-353 and Phe-397, along with addition of negative charge or removal of positive charge near one of the vanadate oxygens, is very important. Lys-353 and Phe-397 were previously assigned to be essential in peroxide activation and halide binding. Analysis of the catalytic parameters of the mutant vanadium bromoperoxidase from the seaweed Ascophyllum nodosum also adds fuel to the discussion regarding factors governing the halide specificity of vanadium haloperoxidases. This study presents the first example of directed evolution of a vanadium enzyme.


Assuntos
Ascomicetos/enzimologia , Cloreto Peroxidase/genética , Cloreto Peroxidase/metabolismo , Evolução Molecular Direcionada , Ascophyllum/enzimologia , Bromo/metabolismo , Catálise , Escherichia coli/genética , Halogênios/metabolismo , Concentração de Íons de Hidrogênio , Oxirredução
14.
Biochemistry ; 44(21): 7879-84, 2005 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-15910002

RESUMO

The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubulin and various other GTPases in eukaryotic cells. We have designed a novel inhibitor of FtsZ polymerization based on the structure of the natural substrate GTP. The inhibitory activity of 8-bromoguanosine 5'-triphosphate (BrGTP) was characterized by a coupled assay, which allows simultaneous detection of the extent of polymerization (via light scattering) and GTPase activity (via release of inorganic phosphate). We found that BrGTP acts as a competitive inhibitor of both FtsZ polymerization and GTPase activity with a Ki for GTPase activity of 31.8 +/- 4.1 microM. The observation that BrGTP seems not to inhibit tubulin assembly suggests a structural difference of the GTP-binding pockets of FtsZ and tubulin.


Assuntos
Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/metabolismo , Inibidores Enzimáticos/síntese química , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/síntese química , Antibacterianos/metabolismo , Proteínas de Bactérias/ultraestrutura , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Proteínas do Citoesqueleto/ultraestrutura , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Luz , Espalhamento de Radiação
15.
J Bacteriol ; 187(9): 3122-32, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15838039

RESUMO

The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).


Assuntos
Cupriavidus necator/enzimologia , Hidrogenase/química , Hidrogenase/metabolismo , NADP/metabolismo , Subunidades Proteicas/química , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Sequência Conservada , Complexo I de Transporte de Elétrons/química , Ativação Enzimática , Estabilidade Enzimática , Dados de Sequência Molecular , Peso Molecular , Família Multigênica , Ligação Proteica , Homologia de Sequência de Aminoácidos
16.
Org Biomol Chem ; 1(16): 2833-9, 2003 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-12968332

RESUMO

Nonspecific acid phosphatases share a conserved active site with mammalian glucose-6-phosphatases (G6Pase). In this work we examined the kinetics of the phosphorylation of glucose and dephosphorylation of glucose-6-phosphate (G6P) catalysed by the acid phosphatases from Shigella flexneri (PhoN-Sf) and Salmonella enterica (PhoN-Se). PhoN-Sf is able to phosphorylate glucose regiospecifically to G6P, glucose-1-phosphate is not formed. The K(m) for glucose using pyrophosphate (PPi) as a phosphate donor is 5.3 mM at pH 6.0. This value is not significantly affected by pH in the pH region 4-6. The K(m) value for G6P by contrast is much lower (0.02 mM). Our experiments show these bacterial acid phosphatases form a good model for G6Pase. We also studied the phosphorylation of inosine to inosine monophosphate (IMP) using PPi as the phosphate donor. PhoN-Sf regiospecifically phosphorylates inosine to inosine-5'-monophosphate whereas PhoN-Se produces both 5'IMP and 3'IMP. The data show that during catalysis an activated phospho-enzyme intermediate is formed that is able to transfer its phosphate group to water, glucose or inosine. A general mechanism is presented of the phosphorylation and dephosphorylation reaction catalysed by the acid phosphatases. Considering the nature of the substrates that are phosphorylated it is likely that this class of enzyme is able to phosphorylate a wide range of hydroxy compounds.


Assuntos
Fosfatase Ácida/metabolismo , Glucose-6-Fosfato/metabolismo , Glucose/metabolismo , Inosina Monofosfato/metabolismo , Inosina/metabolismo , Organofosfatos/metabolismo , Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/genética , Ascophyllum/enzimologia , Inibidores Enzimáticos/farmacologia , Glucose/farmacologia , Cinética , Peroxidases/metabolismo , Fosforilação , Fosfotransferases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella enterica/enzimologia , Shigella flexneri/enzimologia , Estereoisomerismo
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