RESUMO
The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme.
Assuntos
Desidrogenases de Carboidrato/química , Desidrogenases de Carboidrato/metabolismo , Fenilalanina/metabolismo , Trametes/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Ativação Enzimática , Estabilidade Enzimática , Peróxido de Hidrogênio/metabolismo , Cinética , Espectrometria de Massas , Metionina/metabolismo , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Oxirredução , Peptídeos/química , Especificidade por SubstratoRESUMO
The flavin-dependent homotetrameric enzyme pyranose 2-oxidase (P2O) is found mostly, but not exclusively, in lignocellulose-degrading fungi where it catalyzes the oxidation of ß-d-glucose to the corresponding 2-keto sugar concomitantly with hydrogen peroxide formation during lignin solubilization. Here, we present crystal structures of P2O from the efficient lignocellulolytic basidiomycete Phanerochaete chrysosporium. Structures were determined of wild-type PcP2O from the natural fungal source, and two variants of recombinant full-length PcP2O, both in complex with the slow substrate 3-deoxy-3-fluoro-ß-d-glucose. The active sites in PcP2O and P2O from Trametes multicolor (TmP2O) are highly conserved with identical substrate binding. Our structural analysis suggests that the 17 °C higher melting temperature of PcP2O compared to TmP2O is due to an increased number of intersubunit salt bridges. The structure of recombinant PcP2O expressed with its natural N-terminal sequence, including a proposed propeptide segment, reveals that the first five residues of the propeptide intercalate at the interface between A and B subunits to form stabilizing, mainly hydrophobic, interactions. In the structure of mature PcP2O purified from the natural source, the propeptide segment in subunit A has been replaced by a nearby loop in the B subunit. We propose that the propeptide in subunit A stabilizes the A/B interface of essential dimers in the homotetramer and that, upon maturation, it is replaced by the loop in the B subunit to form the mature subunit interface. This would imply that the propeptide segment of PcP2O acts as an intramolecular chaperone for oligomerization at the A/B interface of the essential dimer.
RESUMO
The lnbA gene of Lactococcus lactis ssp. lactis IL1403 encodes a polypeptide with similarity to lacto-N-biosidases and N-acetyl-ß-D-hexosaminidases. The gene was cloned into the expression vector pET-21d and overexpressed in Escherichia coli BL21* (DE3). The recombinant purified enzyme (LnbA) was a monomer with a molecular weight of approximately 37 kDa. Studies with chromogenic substrates including p-nitrophenyl N-acetyl-ß-D-glucosamine (pNP-GlcNAc) and p-nitrophenyl N-acetyl-ß-D-galactosamine (pNP-GalNAc) showed that the enzyme had both N-acetyl-ß-D-glucosaminidase and N-acetyl-ß-D-galactosaminidase activity, thus indicating that the enzyme is an N-acetyl-ß-D-hexosaminidase. K(m) and k(cat) for pNP-GlcNAc were 2.56 mM and 26.7 s(-1), respectively, whereas kinetic parameters for pNP-GalNAc could not be determined due to the K(m) being very high (>10 mM). The optimal temperature and pH of the enzyme were 37 °C and 5.5, respectively, for both substrates. The half-life of activity at 37 °C and pH 6.0 was 53 h, but activity was completely abolished after 30 min at 50 °C, meaning that the enzyme has relatively low temperature stability. The enzyme was stable in the pH 5.5-8 range and was unstable at pH below 5.5. Studies with natural substrates showed hydrolytic activity on chito-oligosaccharides but not on colloidal chitin or chitosan. Transglycosylation products were not detected. In all, the data suggest that LnbA's role may be to degrade chito-oligosaccharides that are produced by the previously described chitinolytic system of L. lactis.
Assuntos
Expressão Gênica , Lactococcus lactis/enzimologia , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/metabolismo , Quitina/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Cinética , Lactococcus lactis/genética , Oligossacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura , beta-N-Acetil-Hexosaminidases/químicaRESUMO
Sugar oxidoreductases such as cellobiose dehydrogenase or pyranose oxidase are widespread enzymes among fungi, whose biological function is largely speculative. We investigated a similar gene family in the mushroom Agaricus meleagris and its expression under various conditions. Three genes (named pdh1, pdh2 and pdh3) putatively encoding pyranose dehydrogenases were isolated. All three genes displayed a conserved structure and organization, and the respective cDNAs contained ORFs translating into polypeptides of 602 or 600 amino acids. The N-terminal sections of all three genes encode putative signal peptides consistent with the enzymes extracellular secretion. We cultivated the fungus on different carbon sources and analyzed the mRNA levels of all three genes over a period of several weeks using real-time RT-PCR. The glyceraldehyde-3-phosphate dehydrogenase gene from A. meleagris was also isolated and served as reference gene. pdh2 and pdh3 are essentially transcribed constitutively, whereas pdh1 expression is upregulated upon exhaustion of the carbon source; pdh1 appears to be additionally regulated under conditions of oxygen limitation. These data are consistent with an assumed role in lignocellulose degradation.
Assuntos
Agaricus/genética , Desidrogenases de Carboidrato/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Celulose/metabolismo , Clonagem Molecular , Proteínas Fúngicas/genética , Lignina/metabolismo , Proteínas de Membrana/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/análiseRESUMO
Pyranose dehydrogenase (PDH) of the mushroom Agaricus meleagris was purified from mycelial culture media to substantial homogeneity using ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric polypeptide with a molecular mass of 66,547Da as determined by matrix-assisted laser desorption/ionisation mass spectrometry containing approximately 7% carbohydrate and covalently bound flavin adenine dinucleotide. The enzyme exhibited a broad sugar substrate tolerance, oxidizing different aldopyranoses to the corresponding C-2 or C-2,3 (di)dehydro sugars. Preferred electron donors with the highest k(cat)/K(m) values were major sugar constituents of cellulose and hemicellulose, namely d-glucose, D-galactose, l-arabinose, D-xylose and cellobiose. This indicates a possible physiological role of the enzyme in lignocellulose breakdown. PDH showed no detectable activity with oxygen, and its reactivity towards electron acceptors was limited to various substituted benzoquinones and complexed metal ions, with the ferricenium ion and the benzoquinone imine 2,6-dichloroindophenole displaying the highest k(cat)/K(m). The enzyme catalyzed in up to 95% yields the regiospecific conversion of D-galactose to 2-dehydro-D-galactose, an intermediate in a possible biotechnologically interesting process for redox isomerization of D-galactose to the prebiotic sugar D-tagatose.