RESUMO
The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na(+) and Ca(2+) ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and beta-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.
Assuntos
Bactérias Aeróbias Gram-Negativas/enzimologia , Xilosidases/química , Xilosidases/metabolismo , Sequência de Bases , Sítios de Ligação , Metabolismo dos Carboidratos , Domínio Catalítico , Clonagem Molecular , Primers do DNA/genética , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Bactérias Aeróbias Gram-Negativas/genética , Temperatura Alta , Espectroscopia de Ressonância Magnética , Desnaturação Proteica , Especificidade por Substrato , Termodinâmica , Xilano Endo-1,3-beta-Xilosidase , Xilosidases/genéticaRESUMO
The xynl encoded 5 domain xylanase from the thermophilic bacterium Rhodothermus marinus binds specifically to xylan, beta-glucan and amorphous but not crystalline cellulose. Our results show that the binding is mediated by the full length xylanase, but not by the catalytic domain only. Based on similarities concerning both predicted secondary structure and binding specificity found with one cellulose binding domain of CenC from Cellulomonas fimi, we suggest that the binding is mediated by the two N-terminally repeated domains.
Assuntos
Bacteroidetes/enzimologia , Celulose/metabolismo , Glucanos/metabolismo , Xilanos/metabolismo , Xilosidases/metabolismo , Sequência de Aminoácidos , Bacteroidetes/genética , Domínio Catalítico , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Ligação Proteica , Sinais Direcionadores de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Xilano Endo-1,3-beta-Xilosidase , Xilosidases/química , Xilosidases/genéticaRESUMO
The gene (xyn1) encoding a Rhodothermus marinus xylanase has been cloned and expressed in Escherichia coli. The gene comprises 5 different domains in an unusual combination. The cellulose binding domains (CBDs) encoded by xyn1 are repeated in tandem at the N-terminus and show similarity with the CBD family IV. The xyn1-gene is the first example encoding a CBD family IV in combination with a xylan hydrolyzing catalytic domain of the glycosyl hydrolase family 10.
Assuntos
Genes Bacterianos , Bactérias Aeróbias Gram-Negativas/enzimologia , Xilosidases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Endo-1,4-beta-Xilanases , Bactérias Aeróbias Gram-Negativas/genética , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
A cDNA sequence encoding a neutral horseradish peroxidase, HRP-n, was found to be growth inhibiting and under certain circumstances also toxic to Escherichia coli upon expression. The growth inhibiting and toxic activity was identified to a sequence of 192 nucleotides which encode the first deduced 64 N-terminal amino acids of the total 299 amino acids in the mature, neutral horseradish peroxidase. The sequence makes part of the active site of the enzyme and is very conserved among peroxidases. The cDNA sequence was cloned in the heat inducible expression vector pJLA603. The toxic effect was mediated by the produced polypeptide since no growth inhibiting or toxic activity was observed when the cDNA sequence was induced after ligation in a wrong reading frame. Generation of oxygen radicals was not the mechanism behind the toxicity, since the effect was observed even after induction of the complete HRP-n sequence or the identified toxic sequence under anaerobic conditions.
Assuntos
Escherichia coli/genética , Peroxidase do Rábano Silvestre/genética , Sequência de Aminoácidos , Sequência de Bases , Biotecnologia , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica , Genes de Plantas , Vetores Genéticos , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas Recombinantes/genéticaRESUMO
A cDNA clone encoding a horseradish (Armoracia rusticana) peroxidase has been isolated and characterized. The cDNA contains 1378 nucleotides excluding the poly(A) tail and the deduced protein contains 327 amino acids which includes a 28 amino acid leader sequence. The predicted amino acid sequence is nine amino acids shorter than the major isoenzyme belonging to the horseradish peroxidase C group (HRP-C) and the sequence shows 53.7% identity with this isoenzyme. The described clone encodes nine cysteines of which eight correspond well with the cysteines found in HRP-C. Five potential N-glycosylation sites with the general sequence Asn-X-Thr/Ser are present in the deduced sequence. Compared to the earlier described HRP-C this is three glycosylation sites less. The shorter sequence and fewer N-glycosylation sites give the native isoenzyme a molecular weight of several thousands less than the horseradish peroxidase C isoenzymes. Comparison with the net charge value of HRP-C indicates that the described cDNA clone encodes a peroxidase which has either the same or a slightly less basic pI value, depending on whether the encoded protein is N-terminally blocked or not. This excludes the possibility that HRP-n could belong to either the HRP-A, -D or -E groups. The low sequence identity (53.7%) with HRP-C indicates that the described clone does not belong to the HRP-C isoenzyme group and comparison of the total amino acid composition with the HRP-B group does not place the described clone within this isoenzyme group. Our conclusion is that the described cDNA clone encodes a neutral horseradish peroxidase which belongs to a new, not earlier described, horseradish peroxidase group.
