Modulating O2 Reactivity in a Fungal Flavoenzyme

Hernández-Ortega, Aitor; Lucas, Fátima; Ferreira, Patricia; Medina, Milagros; Guallar, Vı́ctor; Martínez, Ángel T.

doi:10.1074/jbc.m111.282467

Cited by 50 publications

(36 citation statements)

References 52 publications

(59 reference statements)

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“…3). The M-I segment includes the pre␣proK (to evolve the chimeric leader for secretion) plus a region of the mature AAO (Ala1-Val109) that contains the FAD-binding domain at its N terminus, along with several structural determinants in the nearby substrate access channel: Val54, Pro55, His91, Tyr92, Pro79, and Val90 (4,6,30). The M-II segment contains the catalytic pocket, including the His546 involved in substrate positioning, the catalytic base His502, and the aromatic residues Phe397 and Phe501.…”

Section: Resultsmentioning

confidence: 99%

“…The past few years have witnessed an intense effort to discern the basis and mechanism of action underlying AAO catalysis (3)(4)(5)(6)(7)(8)(9)(10). The AAO catalytic cycle involves dehydrogenative oxidation mediated by two half-reactions: (i) the reductive half-reaction in which the donor alcohol is two-electron oxidized by the FAD cofactor, the latter receiving one of the alcohol's ␣-Hs through a hydride transfer process that yields the aldehyde product and the reduced flavin, and (ii) the oxidative half-reaction, in which O 2 is two-electron reduced by the FAD, releasing H 2 O 2 and the reoxidized flavin (5).…”

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confidence: 99%

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Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

Viña‐Gonzalez

González-Pérez

Ferreira

et al. 2015

Appl Environ Microbiol

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c Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H 2 O 2 during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO is an attractive candidate for studies into organic synthesis and synthetic biology, and yet the lack of suitable heterologous expression systems has precluded its engineering by directed evolution. In this study, the native signal sequence of AAO from Pleurotus eryngii was replaced by those of the mating ␣-factor and the K 1 killer toxin, as well as different chimeras of both prepro-leaders in order to drive secretion in Saccharomyces cerevisiae. The secretion of these AAO constructs increased in the following order: prepro␣-AAO > pre␣proK-AAO > preKpro␣-AAO > preproK-AAO. The chimeric pre␣proK-AAO was subjected to focused-directed evolution with the aid of a dual screening assay based on the Fenton reaction. Random mutagenesis and DNA recombination was concentrated on two protein segments (Met[␣1]-Val109 and Phe392-Gln566), and an array of improved variants was identified, among which the FX7 mutant (harboring the H91N mutation) showed a dramatic 96-fold improvement in total activity with secretion levels of 2 mg/liter. Analysis of the N-terminal sequence of the FX7 variant confirmed the correct processing of the pre␣proK hybrid peptide by the KEX2 protease. FX7 showed higher stability in terms of pH and temperature, whereas the pH activity profiles and the kinetic parameters were maintained. The Asn91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily, except for P. eryngii and P. pulmonarius AAO. The in vitro involution of the enzyme by restoring the consensus ancestor Asn91 promoted AAO expression and stability.A ryl-alcohol oxidase (AAO; EC 1.1.3.7) is a flavoenzyme of the GMC (glucose-methanol-choline) oxidoreductase superfamily, the members of which share a N-terminal FAD-binding domain containing the canonical ADP-binding motif. Secreted by several white-rot fungi, this monomeric flavoprotein plays an essential role in natural lignin degradation (1). Accordingly, AAO oxidizes lignin-derived compounds and aromatic fungal metabolites, releasing H 2 O 2 that is required by ligninolytic peroxidases to attack the plant cell wall (2). Moreover, the H 2 O 2 produced by AAO is an efficient vehicle to generate highly reactive hydroxyl radicals through the Fenton reaction (Fe 2ϩ ϩ H 2 O 2 ¡ OH˙ϩ OH Ϫ ϩ Fe 3ϩ ), such that OH˙can act as a diffusible electron carrier to depolymerize plant polymers. AAO oxidizes a variety of aromatic benzyl (and some aliphatic polyunsaturated) alcohols to the corresponding aldehydes. In addition, AAO participates in the oxidation of aromatic aldehydes to the corresponding acids and also has activity on furfural derivatives (3).The past few years have witnessed an intense effort to discern the basis and mechanism of action underlying AAO catalysis (3-10). The AAO catalytic cycle involves dehydroge...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

Viña‐Gonzalez

González-Pérez

Ferreira

et al. 2015

Appl Environ Microbiol

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“…Reduced oxygen reactivity is a desirable property for some applications because of reduced The reactivity of flavoproteins with oxygen is of considerable scientific interest, and the determinants of oxygen activation and reactivity are the subject of numerous studies [39][40][41][42][43][44]. The consensus is that there is no single parameter (like spatial accessibility, charge, hydrophobicity, vicinity of particular amino acid residues etc.)…”

Section: Discussionmentioning

confidence: 99%

“…Channels leading from the surface to the active site may affect oxidase reactivity [37,38]. Altering the oxygen reactivity is of considerable interest as shown in recent reviews [15,28] and works of Krondorfer et al [39], Sygmund et al [40], Leferink et al [41], and others [42][43][44].…”

Section: Introductionmentioning

confidence: 99%

Engineering of pyranose 2-oxidase for modified oxygen reactivity

2014

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“…4). As previously described for P. eryngii AAO, a funnel-shaped channel constricted by a bottleneck, involving three aromatic residue side chains (Y92, F397, and F501), connects the active-site cavity to the solvent and provides access for O 2 and reducing substrates to the buried active site (43). Residues involved in the formation of this access channel are drastically different from those in the P. eryngii crystal structure ( Fig.…”

Section: Phylogenetic Analysis and Sequence Analysismentioning

confidence: 99%

Activities of Secreted Aryl Alcohol Quinone Oxidoreductases from Pycnoporus cinnabarinus Provide Insights into Fungal Degradation of Plant Biomass

Mathieu

Piumi

Valli

et al. 2016

Appl Environ Microbiol

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Auxiliary activities family 3 subfamily 2 (AA3_2) from the CAZy database comprises various functions related to ligninolytic enzymes, such as fungal aryl alcohol oxidases (AAO) and glucose oxidases, both of which are flavoenzymes. The recent study of the Pycnoporus cinnabarinus CIRM BRFM 137 genome combined with its secretome revealed that four AA3_2 enzymes are secreted during biomass degradation. One of these AA3_2 enzymes, scf184803.g17, has recently been produced heterologously in Aspergillus niger. Based on the enzyme's activity and specificity, it was assigned to the glucose dehydrogenases (P. cinnabarinus GDH [PcGDH]). Here, we analyze the distribution of the other three AA3_2 enzymes (scf185002.g8, scf184611.g7, and scf184746.g13) to assess their putative functions. These proteins showed the highest homology with aryl alcohol oxidase from Pleurotus eryngii. Biochemical characterization demonstrated that they were also flavoenzymes harboring flavin adenine dinucleotide (FAD) as a cofactor and able to oxidize a wide variety of phenolic and nonphenolic aryl alcohols and one aliphatic polyunsaturated primary alcohol. Though presenting homology with fungal AAOs, these enzymes exhibited greater efficiency in reducing electron acceptors (quinones and one artificial acceptor) than molecular oxygen and so were defined as aryl-alcohol: quinone oxidoreductases (AAQOs) with two enzymes possessing residual oxidase activity (PcAAQO2 and PcAAQO3). Structural comparison of PcAAQO homology models with P. eryngii AAO demonstrated a wider substrate access channel connecting the active-site cavity to the solvent, explaining the absence of activity with molecular oxygen. Finally, the ability of PcAAQOs to reduce radical intermediates generated by laccase from P. cinnabarinus was demonstrated, shedding light on the ligninolytic system of this fungus.A uxiliary activities (AA) are a widespread group of catalytic modules involved in plant cell wall degradation. They were first introduced in 2013 into the carbohydrate-active enzymes (CAZy [www.cazy.org]) database (1). This AA class encompasses and extends an earlier classification dedicated to fungal ligninolytic enzymes and comprises 10 families, some of which have subfamilies (2). The AA family group enzymes possess the potential ability to help the original carbohydrate-active enzymes gain access to the carbohydrates embedded in the plant cell wall. Lignin, the main noncarbohydrate structural component of plant cell walls, consists of an intricate network of aromatic compounds forming a strong, hydrophobic, insoluble barrier closing access to the carbohydrates. Microorganisms have therefore set up different strategies to either modify or degrade lignin as a first step to gain access to the carbohydrate moiety and convert it into an energy source (3).In this new AA classification, family AA3 belongs to the glucose-methanol-choline (GMC) oxidoreductase family first defined by Cavener (4). AA3 enzymes are flavoproteins containing a flavin adenine dinucleotide (FAD)-binding domai...

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Modulating O2 Reactivity in a Fungal Flavoenzyme

Cited by 50 publications

References 52 publications

Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

Engineering of pyranose 2-oxidase for modified oxygen reactivity

Activities of Secreted Aryl Alcohol Quinone Oxidoreductases from Pycnoporus cinnabarinus Provide Insights into Fungal Degradation of Plant Biomass

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