A survey of genes encoding H2O2-producing GMC oxidoreductases in 10 Polyporales genomes

15 p.-4 fig.-1 tab.The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) recently were sequenced to expand our knowledge on the diversity and distribution of genes involved in degradation of plant polymers in this Basidiomycota order, which includes most wood-rotting fungi. Oxidases, including members of the glucose-methanol-choline (GMC) oxidoreductase superfamily, play a central role in the above degradative process because they generate extracellular H2O2 acting as the ultimate oxidizer in both white-rot and brown-rot decay. The survey was completed by analyzing the GMC genes in the available genomes of seven more species to cover the four Polyporales clades. First, an in silico search for sequences encoding members of the aryl-alcohol oxidase, glucose oxidase, methanol oxidase, pyranose oxidase, cellobiose dehydrogenase and pyranose dehydrogenase families was performed. The curated sequences were subjected to an analysis of their evolutionary relationships,followed by estimation of gene duplication/ reduction history during fungal evolution. Second,the molecular structures of the near one hundred GMC oxidoreductases identified were modeled to gain insight into their structural variation and expected catalytic properties. In contrast to ligninolytic peroxidases,whose genes are present in all white-rot Polyporales genomes and absent from those of brown-rot species, the H2O generating oxidases are widely distributed in both fungal types. This indicates that the GMC oxidases provide H2O2 for both ligninolytic peroxidase activity (in white-rot decay) and Fenton attack on cellulose (in brown-rot decay), after the transition between both decay patterns in Polyporales occurred.This work was supported by the INDOX (www.indoxproject.eu; KBBE-2013-7-613549) European project and by the Spanish HIPOP (BIO2011-26694) project. The work conducted by the U.S. Department of Energy Joint Genome Institute was supported by the Office of Science of the U.S. Department of Energy under Contract DE-AC02-05CH11231, in the frame of the JGI Saprotrophic Agaricomycotina project coordinated by D.S. Hibbett (Clark University, USA).Peer reviewe

A survey of genes encoding H2O2-producing GMC oxidoreductases in 10 Polyporales genomes

The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) recently were sequenced to expand our knowledge on the diversity and distribution of genes involved in degradation of plant polymers in this Basidiomycota order, which includes most wood-rotting fungi. Oxidases, including members of the glucose-methanol-choline (GMC) oxidoreductase superfamily, play a central role in the above degradative process because they generate extracellular H2O2 acting as the ultimate oxidizer in both white-rot and brown-rot decay. The survey was completed by analyzing the GMC genes in the available genomes of seven more species to cover the four Polyporales clades. First, an in silico search for sequences encoding members of the aryl-alcohol oxidase, glucose oxidase, methanol oxidase, pyranose oxidase, cellobiose dehydrogenase and pyranose dehydrogenase families was performed. The curated sequences were subjected to an analysis of their evolutionary relationships, followed by estimation of gene duplication/ reduction history during fungal evolution. Second, the molecular structures of the near one hundred GMC oxidoreductases identified were modeled to gain insight into their structural variation and expected catalytic properties. In contrast to ligninolytic peroxidases, whose genes are present in all white-rot Polyporales genomes and absent from those of brown-rot species, the H2O2-generating oxidases are widely distributed in both fungal types. This indicates that the GMC oxidases provide H2O2 for both ligninolytic peroxidase activity (in white-rot decay) and Fenton attack on cellulose (in brown-rot decay), after the transition between both decay patterns in Polyporales occurred

On the Catalytic Roles of HIS351, ASN510, and HIS466 in Choline Oxidase and the Kinetic Mechanism of Pyranose 2-Oxidase

Choline oxidase (E.C. 1.1.3.17) from Arthrobacter globiformis catalyzes the four-electron oxidation of choline to glycine betaine (N,N,N-trimethylglycine) via two sequential, FAD-dependent reactions in which betaine aldehyde is formed as an enzyme-bound intermediate. In each oxidative half-reaction, molecular oxygen acts as electron acceptor and is converted into hydrogen peroxide. Biochemical, structural, and mechanistic studies on the wild-type and a number of mutant variants of choline oxidase have recently been carried out, allowing for the depiction of the mechanism of alcohol oxidation catalyzed by the enzyme. Catalysis by choline oxidase is initiated by the removal of the hydroxyl proton of alcohol substrate by a catalytic base in the enzyme-substrate complex, yielding the formation of the alkoxide species. In this dissertation, the roles of His351 and conserved His466 were investigated. The results presented demonstrate that His351 is involved in the stabilization of the transition state for the hydride transfer reaction and contributes to substrate binding. His466 is likely to be a catalytic base in choline oxidase due to its dramatic effect on enzymatic activity. Comparison of choline oxidase and other enzymes within its superfamily reveals the presence of a conserved His-Asn pair within the active site of enzymes. Therefore, the role of the conserved Asn510 in choline oxidase was examined in this study. The results presented here establish the importance of Asn510 in both the reductive and oxidative half-reactions. The lost of ability to form a hydrogen bond interaction between the side chain at position 510 with neighboring residues such as His466 resulted in a change from stepwise to concerted mechanism for the cleavages of OH and CH bonds of choline, as seen in the Asn510Ala mutant. Finally, the steady-state kinetic mechanism of pyranose 2-oxidase in the pH range from 5.5 to 8.5 was investigated. It was found that pH exerts significant effects on enzyme mechanism. This study has established the involvement of the residues in the initiation of enzyme catalysis and the stabilization of the alkoxide intermediate in choline oxidase. In addition, this work demonstrates the first instance in which the kinetic mechanism of a flavin-dependent oxidase is governed by pH

On the Mechanistic Roles of the Protein Positive Charge Close to the N(1)Flavin Locus in Choline Oxidase

Choline oxidase catalyzes the oxidation of choline to glycine betaine. This reaction is of considerable medical and biotechnological applications, because the accumulation of glycine betaine in the cytoplasm of many plants and human pathogens enables them to counteract hyperosmotic environments. In this respect, the study of choline oxidase has potential for the development of a therapeutic agent that can specifically inhibit the formation of glycine betaine, and therefore render pathogens more susceptible to conventional treatment. The study of choline oxidase has also potential for the improvement of the stress resistance of plant by introducing an efficient biosynthetic pathway for glycine betaine in genetically engineered economically relevant crop plant. In this study, codA gene encoding for choline oxidase was cloned. The cloned gene was then used to express and purify the wild-type enzyme as well as to prepare selected mutant forms of choline oxidase. In all cases, the resulting enzymes were purified to high levels, allowing for detailed characterizations. The biophysical and biochemical analyses of choline oxidase variants in which the positively charged residue close to the flavin N(1) locus (His466) was removed (H466A) or reversed (H466D) suggest that in choline oxidase, His466 modulates the electrophilicity of the bound flavin and the polarity of the active site, and contributes to the flavinylation process of the covalently bound FAD as well as to the stabilization of the negative charges in the active site. Biochemical, structural, and mechanistic relevant properties of selected flavoproteins with special attention to flavoprotein oxidases, as well as the biotechnological and medical relevance of choline oxidase, are presented in Chapter I. Chapter II summarizes all the experimental techniques used in this study. Chapter III-VII illustrate my studies on choline oxidase, including cloning, expression, purification and preliminary characterizations (Chapter III), spectroscopic and steady state kinetics (Chapter IV), the catalytic roles of His466 and the effects of reversing the protein positive charge close to the flavin N(1) locus (Chapter V and VI), and the roles of His310 with a special attention to its involvement in a proton-transfer network (Chapter VII). Chapter VIII presents a general discussion of the data presented

Analysis of the structure and substrate scope of chitooligosaccharide oxidase reveals high affinity for C2-modified glucosamines

Chitooligosaccharide oxidase (ChitO) is a fungal carbohydrate oxidase containing a bicovalently bound FAD cofactor. The enzyme is known to catalyse the oxidation of chitooligosaccharides, oligomers of N-acetylated glucosamines derived from chitin degradation. In this study, the unique substrate acceptance was explored by testing a range of N-acetyl-D-glucosamine derivatives, revealing that ChitO preferentially accepts carbohydrates with a hydrophobic group attached to C2. The enzyme also accepts streptozotocin, a natural product used to treat tumours. Elucidation of the crystal structure provides an explanation for the high affinity towards C2-decorated glucosamines: the active site has a secondary binding pocket that accommodates groups attached at C2. Docking simulations are fully in line with the observed substrate preference. This work expands the knowledge on this versatile enzyme

Characterization of three pyranose dehydrogenase isoforms from the litter-decomposing basidiomycete Leucoagaricus meleagris (syn. Agaricus meleagris)

Nicht verfügbarMultigenicity is commonly found in fungal enzyme systems, with the purpose of functional compensation upon deficiency of one of its members or leading to enzyme isoforms with new functionalities through gene diversification. Three genes of the flavin-dependent glucosemethanolcholine (GMC) oxidoreductase pyranose dehydrogenase (AmPDH) were previously identified in the litter-degrading fungus Agaricus (Leucoagaricus) meleagris, of which only AmPDH1 was successfully expressed and characterized. The aim of this work was to study the biophysical and biochemical properties of AmPDH2 and AmPDH3 and compare them with those of AmPDH1. AmPDH1, AmPDH2 and AmPDH3 showed negligible oxygen reactivity and possess a covalently tethered FAD cofactor. All three isoforms can oxidise a range of different monosaccarides and oligosaccharides including glucose, mannose, galactose and xylose, which are the main constituent sugars of cellulose and hemicelluloses, and judging from the apparent steady-state kinetics determined for these sugars, the three isoforms do not show significant differences pertaining to their reaction with sugar substrates. They oxidize glucose both at C2 and C3 and upon prolonged reaction C2 and C3 double-oxidized glucose is obtained, confirming that the A. meleagris genes pdh2 (AY753308.1) and pdh3 (DQ117577.1) indeed encode CAZy class AA3_2 pyranose dehydrogenases. While reactivity with electron donor substrates was comparable for the three AmPDH isoforms, their kinetic properties differed significantly for the model electron acceptor substrates tested, a radical (the 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] cation radical), a quinone (benzoquinone) and a complexed iron ion (the ferricenium ion). Thus, a possible explanation for this PDH multiplicity in A. meleagris could be that different isoforms react preferentially with structurally different electron acceptors in vivo.(VLID)192910

Structural Elucidation and Engineering of a Bacterial Carbohydrate Oxidase

Flavin-dependent carbohydrate oxidases are valuable tools in biotechnological applications due to their high selectivity in the oxidation of carbohydrates. In this study, we report the biochemical and structural characterization of a recently discovered carbohydrate oxidase from the bacterium Ralstonia solanacearum, which is a member of the vanillyl alcohol oxidase flavoprotein family. Due to its exceptionally high activity toward N-acetyl-d-galactosamine and N-acetyl-d-glucosamine, the enzyme was named N-acetyl-glucosamine oxidase (NagOx). In contrast to most known (fungal) carbohydrate oxidases, NagOx could be overexpressed in a bacterial host, which facilitated detailed biochemical and enzyme engineering studies. Steady state kinetic analyses revealed that non-acetylated hexoses were also accepted as substrates albeit with lower efficiency. Upon determination of the crystal structure, structural insights into NagOx were obtained. A large cavity containing a bicovalently bound FAD, tethered via histidyl and cysteinyl linkages, was observed. Substrate docking highlighted how a single residue (Leu251) plays a key role in the accommodation of N-acetylated sugars in the active site. Upon replacement of Leu251 (L251R mutant), an enzyme variant was generated with a drastically modified substrate acceptance profile, tuned toward non-N-acetylated monosaccharides and disaccharides. Furthermore, the activity toward bulkier substrates such as the trisaccharide maltotriose was introduced by this mutation. Due to its advantage of being overexpressed in a bacterial host, NagOx can be considered a promising alternative engineerable biocatalyst for selective oxidation of monosaccharides and oligosaccharides.</p

The vast repertoire of carbohydrate oxidases:An overview

Carbohydrates are widely abundant molecules present in a variety of forms. For their biosynthesis and modification, nature has evolved a plethora of carbohydrate-acting enzymes. Many of these enzymes are of particular interest for biotechnological applications, where they can be used as biocatalysts or biosensors. Among the enzymes catalysing conversions of carbohydrates are the carbohydrate oxidases. These oxidative enzymes belong to different structural families and use different cofactors to perform the oxidation reaction of CH-OH bonds in carbohydrates. The variety of carbohydrate oxidases available in nature reflects their specificity towards different sugars and selectivity of the oxidation site. Thanks to their properties, carbohydrate oxidases have received a lot of attention in basic and applied research, such that nowadays their role in biotechnological processes is of paramount importance. In this review we provide an overview of the available knowledge concerning the known carbohydrate oxidases. The oxidases are first classified according to their structural features. After a description on their mechanism of action, substrate acceptance and characterisation, we report on the engineering of the different carbohydrate oxidases to enhance their employment in biocatalysis and biotechnology. In the last part of the review we highlight some practical applications for which such enzymes have been exploited

New Gmc oxidoreductases from ligninolytic basydiomycetes: genomic screening, catalytic mechanisms and biotechnological potential

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Bioquímica y Biología Molecular I, leída el 10-05-2017La superfamilia de enzymas glucosa-metanol-colina (GMC) oxidasas/deshidrogenasas se compone de proteínas que contienen FAD filogenéticamente relacionadas entre sí que comparten un plegamiento común que se subdivide en dos dominios diferentes: el de unión a FAD y el de unión a sustrato. Algunas oxidorreductasas fúngicas que pertenecen a esta superfamilia desempeñan la función de enzimas auxiliares en el proceso de degradación de la lignocelulosa. La mayor parte de ellas son oxidorreductasas extracelulares que producen el H2O2 requerido: (i) por las peroxidasas de alto potencial redox para actuar sobre la lignina; o (ii) para desencadenar las reaciones de Fenton, que dan lugar a especies radicales de oxígeno que atacan a la lignocelulosa.El estudio de los genomas de 10 hongos seleccionados del orden Poliporales, los únicos organismos capaces de mineralizar totalmente la lignina, arrojó luz sobre la participación de 5 familias de oxidorreductasas GMC en la descomposición de la lignocelulosa. Estas fueron las glucosa oxidasas (GOX), celobiosa deshidrogenasas (CDH), piranosa 2-oxidasas (P2O), metanol oxidasas (MOX) y aril-alcohol oxidasas (AAO). Los estudios filogenéticos sugieren que estas enzimas comparten un ancestro común cuya diversificación tuvo lugar en una etapa más temprana de la evolución fúngica, que se caracterizó por el escaso número de estos genes GMC. Las AAO y las MOX resultaron ser las oxidorreductasas GMC más abundantes en los genomas seleccionados. El número de genes que codifican para estas proteínas varía ampliamente de un genoma a otro de acuerdo con las características ecofisiológicas de cada hongo...The glucose-methanol-choline oxidase/dehydrogenase (GMC) superfamily of enzymes is composed of FAD-containing, phylogenetically-related proteins that share a common fold subdivided into two different domains: the FAD-binding and substrate-binding ones. Some fungal oxidoreductases belonging to the superfamily play a role as auxiliary enzymes in the lignocellulose-degrading process. Most of them are extracellular oxidoreductases that produce the H2O2 required: (i) by high redox potential peroxidases to act on lignin; or (ii) to trigger Fenton reactions, which give rise to radical oxygen species that attack lignocellulose.Investigation of the genomes of 10 selected Polyporales fungi, the only microorganisms able to completely mineralize lignin, shed light on the involvement of 5 families of GMC oxidoreductases in the lignocellulose decay. These were glucose oxidases (GOX), cellobiose dehydrogenases (CDH), pyranose 2-oxidases (P2O), methanol oxidases (MOX) and aryl-alcohol oxidases (AAO). Phylogenetic studies carried out suggest that they share a common ancestor whose diversification took place at a more ancestral stage of fungal evolution, which is characterized by a low number of these GMC genes. AAO and MOX proved to be the most abundant GMC oxidoreductases present in the selected genomes. The number of genes encoding for these enzymes varies from one genome to another according to the ecophysiology of the fungi. In fact, brown-rot fungi, which do not mineralize lignin but alter its structure to gain access to carbohydrates, lost most of their lignin-degrading enzymes and GMC repertoire, except MOX. These fungi cause the demethoxylation of lignin that releases methanol and relies on the reactivity of radical oxygen species stemming from H2O2. These findings point out the co-evolution of these fungal GMC enzymes with the lignin-degrading peroxidases...Sección Deptal. de Bioquímica y Biología Molecular (Biológicas)Fac. de Ciencias BiológicasTRUEunpu