RESUMEN
ß-Mannanases are involved in the conversion and modification of mannan-based saccharides. Using a retaining mechanism, they can, in addition to hydrolysis, also potentially perform transglycosylation reactions, synthesizing new glyco-conjugates. Transglycosylation has been reported for ß-mannanases in GH5 and GH113. However, although they share the same fold and catalytic mechanism, there may be differences in the enzymes' ability to perform transglycosylation. Three GH5 ß-mannanases from Aspergillus nidulans, AnMan5A, AnMan5B and AnMan5C, which belong to subfamily GH5_7 were studied. Comparative studies, including the GH5_7 TrMan5A from Trichoderma reesei, showed some differences between the enzymes. All the enzymes could perform transglycosylation but AnMan5B stood out in generating comparably higher amounts of transglycosylation products when incubated with manno-oligosaccharides. In addition, AnMan5B did not use alcohols as acceptor, which was also different compared to the other three ß-mannanases. In order to map the preferred binding of manno-oligosaccharides, incubations were performed in H2 (18)O. AnMan5B in contrary to the other enzymes did not generate any (18)O-labelled products. This further supported the idea that AnMan5B potentially prefers to use saccharides as acceptor instead of water. A homology model of AnMan5B showed a non-conserved Trp located in subsite +2, not present in the other studied enzymes. Strong aglycone binding seems to be important for transglycosylation with saccharides. Depending on the application, it is important to select the right enzyme.
Asunto(s)
Aspergillus nidulans/enzimología , beta-Manosidasa/metabolismo , Alcoholes/metabolismo , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Trichoderma/enzimología , Agua/metabolismoRESUMEN
The gene encoding ß-mannanase (EC 3.2.1.78) BaMan26A from the bacterium Bifidobacterium adolescentis (living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed in Escherichia coli without the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 µM (8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K, k(cat) was determined to be 444 s(-1) and K(m) 21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides.
Asunto(s)
Secuencias de Aminoácidos , Bifidobacterium/enzimología , Bifidobacterium/genética , beta-Manosidasa/genética , beta-Manosidasa/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Bacteriana , Sitios de Unión , Gatos , Clonación Molecular , Escherichia coli/genética , Galactosa/análogos & derivados , Expresión Génica , Humanos , Hidrólisis , Cinética , Mananos/metabolismo , Manosa/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
α-Glucuronidases of glycoside hydrolase family 115 of the xylose-fermenting yeast Pichia stipitis and wood-destroying fungus Schizophyllum commune liberate 4-O-methyl-D-glucuronic acid residues from aldouronic acids and glucuronoxylan. The specific activities of both enzymes depended on polymerization degree of the acidic xylooligosaccharides and were inhibited by linear ß-1,4-xylooligosaccharides. These results suggest interaction of the enzyme with several xylopyranosyl residues of the xylan main chain. Using (1)H NMR spectroscopy and reduced aldopentaouronic acid (MeGlcA(3)Xyl(4)-ol) as a substrate, it was found that both enzymes are inverting glycoside hydrolases releasing 4-O-methyl-D-glucuronic acid (MeGlcA) as its ß-anomer.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Pichia/enzimología , Schizophyllum/enzimología , Madera/microbiología , Glicósido Hidrolasas/química , Hidrólisis , Resonancia Magnética Nuclear Biomolecular , Filogenia , Especificidad por SustratoRESUMEN
To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 endo-beta-1,4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant mannans. On the basis of differences in the structures and hydrolytic action patterns of this wild-type (but recombinantly expressed) enzyme and a homologous mannanase from Cellvibrio japonicus, two nonconserved amino acid residues at two distal glycone-binding subsites of the C. fimi enzyme were substituted, Ala323Arg at subsite -2 and Phe325Ala at subsite -3, to achieve inverted mannosyl affinities in the respective subsites, mimicking the Ce. japonicus enzyme that has an Arg providing mannosyl interactions at subsite -2. The X-ray crystal structure of the C. fimi doubly substituted mannanase was determined to 2.35 A resolution and shows that the introduced Arg323 is in a position suitable for hydrogen bonding to mannosyl at subsite -2. We report steady-state enzyme kinetics and hydrolysis-product analyses using anion-exchange chromatography and a novel rapid mass spectrometric profiling method of (18)O-labeled products obtained using H(2)(18)O as a solvent. The results obtained with oligosaccharide substrates show that although the catalytic efficiency (k(cat)/K(m)) is wild-type-like for the engineered enzyme, it has an altered hydrolytic action pattern that stems from promotion of substrate binding at subsite -2 (due to the introduced Arg323) and demotion of it at subsite -3 (to which removal of Phe325 contributed). However, k(cat)/K(m) decreased approximately 1 order of magnitude with polymeric substrates, possibly caused by spatial repositioning of the substrate at subsite -3 and beyond for the engineered enzyme.
Asunto(s)
Cellulomonas/enzimología , Manosa/genética , Manosa/metabolismo , Manosidasas/química , Manosidasas/metabolismo , Ingeniería de Proteínas/métodos , Sustitución de Aminoácidos/genética , Sitios de Unión/genética , Secuencia de Carbohidratos , Cellulomonas/genética , Cellulomonas/metabolismo , Secuencia Conservada , Cristalografía por Rayos X , Hidrólisis , Manosa/química , Manosidasas/genética , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por SustratoRESUMEN
Glucuronoyl esterases are enzymes involved in microbial plant cell-wall degradation. In this study we purified and characterized two recombinant Phanerochaete chrysosporium glucuronoyl esterases, PcGE1 and PcGE2. The catalytic activity of these and previously described glucuronoyl esterases was investigated on new synthetic substrates, methyl esters of uronic acids and their glycosides, prepared by esterification with ethereal diazomethane.The data obtained indicate that the enzymes hydrolyzed efficiently not only esters of 4-O-methyl-D-glucuronic acid, but also methyl esters of D-glucuronic acid carrying a 4-nitrophenyl aglycon. Moreover, the fact that they did not recognize the 4-epimers of these compounds, the D-galacturonic acid derivatives, supports the hypothesis that these carbohydrate esterases attack ester linkages between 4-O-methyl-D-glucuronic acid of glucuronoxylan and lignin alcohols.
Asunto(s)
Esterasas/metabolismo , Phanerochaete/enzimología , Ácidos Urónicos/metabolismo , Esterasas/química , Esterasas/genética , Esterasas/aislamiento & purificación , Cinética , Phanerochaete/genética , Especificidad por SustratoRESUMEN
The plant polysaccharide degradative potential of Aspergillus nidulans was analysed in detail and compared to that of Aspergillus niger and Aspergillus oryzae using a combination of bioinformatics, physiology and transcriptomics. Manual verification indicated that 28.4% of the A. nidulans ORFs analysed in this study do not contain a secretion signal, of which 40% may be secreted through a non-classical method.While significant differences were found between the species in the numbers of ORFs assigned to the relevant CAZy families, no significant difference was observed in growth on polysaccharides. Growth differences were observed between the Aspergilli and Podospora anserina, which has a more different genomic potential for polysaccharide degradation, suggesting that large genomic differences are required to cause growth differences on polysaccharides. Differences were also detected between the Aspergilli in the presence of putative regulatory sequences in the promoters of the ORFs of this study and correlation of the presence of putative XlnR binding sites to induction by xylose was detected for A. niger. These data demonstrate differences at genome content, substrate specificity of the enzymes and gene regulation in these three Aspergilli, which likely reflect their individual adaptation to their natural biotope.
Asunto(s)
Aspergillus nidulans/genética , Aspergillus niger/genética , Aspergillus oryzae/genética , Enzimas/genética , Polisacáridos/metabolismo , Secuencia de Aminoácidos , Aspergillus nidulans/crecimiento & desarrollo , Aspergillus nidulans/metabolismo , Aspergillus niger/crecimiento & desarrollo , Aspergillus niger/metabolismo , Aspergillus oryzae/crecimiento & desarrollo , Aspergillus oryzae/metabolismo , Biología Computacional , Perfilación de la Expresión Génica , Genes Fúngicos , Genoma , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Regiones Promotoras Genéticas/genética , Especificidad por SustratoRESUMEN
Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of "gene sharing," where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst.
Asunto(s)
Carbohidratos/química , Dominio Catalítico/fisiología , Celulasa/metabolismo , Celulosa/metabolismo , Esterasas , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bacteroides/enzimología , Catálisis , Celulasa/química , Celulosa/química , Cellvibrio/enzimología , Esterasas/química , Esterasas/metabolismo , Modelos Moleculares , Polisacáridos/química , Polisacáridos/metabolismoRESUMEN
The mode of action of xylanase A from a phytopathogenic bacterium, Erwinia chrysanthemi, classified in glycoside hydrolase family 5, was investigated on xylooligosaccharides and polysaccharides using TLC, MALDI-TOF MS and enzyme treatment with exoglycosidases. The hydrolytic action of xylanase A was found to be absolutely dependent on the presence of 4-O-methyl-D-glucuronosyl (MeGlcA) side residues in both oligosaccharides and polysaccharides. Neutral linear beta-1,4-xylooligosaccharides and esterified aldouronic acids were resistant towards enzymatic action. Aldouronic acids of the structure MeGlcA(3)Xyl(3) (aldotetraouronic acid), MeGlcA(3)Xyl(4) (aldopentaouronic acid) and MeGlcA(3)Xyl(5) (aldohexaouronic acid) were cleaved with the enzyme to give xylose from the reducing end and products shorter by one xylopyranosyl residue: MeGlcA(2)Xyl(2), MeGlcA(2)Xyl(3) and MeGlcA(2)Xyl(4). As a rule, the enzyme attacked the second glycosidic linkage following the MeGlcA branch towards the reducing end. Depending on the distribution of MeGlcA residues on the glucuronoxylan main chain, the enzyme generated series of shorter and longer aldouronic acids of backbone polymerization degree 3-14, in which the MeGlcA is linked exclusively to the second xylopyranosyl residue from the reducing end. Upon incubation with beta-xylosidase, all acidic hydrolysis products of acidic oligosaccharides and hardwood glucuronoxylans were converted to aldotriouronic acid, MeGlcA(2)Xyl(2). In agreement with this mode of action, xylose and unsubstituted oligosaccharides were essentially absent in the hydrolysates. The E. chrysanthemi xylanase A thus appears to be an excellent biocatalyst for the production of large acidic oligosaccharides from glucuronoxylans as well as an invaluable tool for determination of the distribution of MeGlcA residues along the main chain of this major plant hemicellulose.
Asunto(s)
Proteínas Bacterianas/química , Dickeya chrysanthemi/enzimología , Oligosacáridos de Cadena Ramificada/química , Xilosidasas/química , Proteínas Bacterianas/metabolismo , Catálisis , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Hidrólisis , Cinética , Modelos Moleculares , Estructura Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , Xilanos/química , Xilanos/metabolismo , Xilosidasas/metabolismoRESUMEN
Mode of action of endo-beta-1,4-xylanases (EXs) of glycoside hydrolase families 10 (GH-10) and 11 (GH-11) was examined on various acidic xylooligosaccharides. As expected, none of the enzymes of GH-10 cleaved aldotetraouronic acid (MeGlcA3Xyl3), which is the shortest acidic product of the action of these EXs on glucuronoxylan. Surprisingly, aldopentaouronic acid (MeGlcA3Xyl4) was also not attacked. Only aldohexaouronic acid (MeGlcA3Xyl5) served as a substrate and was cleaved to xylobiose and aldotetraouronic acid. These results suggested that binding of xylopyranosyl residue in the -2 subsite is prerequisite for cleavage of the linkage adjacent to the xylopyranosyl unit carrying MeGlcA. EXs of family GH-11 cleaved neither aldotetraouronic acid, nor aldopentaouronic acid, which is in agreement with their action on glucuronoxylan. Aldohexaouronic acid was cleaved to aldopentaouronic acid and xylobiose without any production of xylose, suggesting that a xylosyl transfer reaction is involved in the degradation of the substrate by EXs of GH-11.