Stereoselective Hydride Transfer by Aryl‐Alcohol Oxidase, a Member of the GMC Superfamily

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

doi:10.1002/cbic.201100709

Cited by 51 publications

(65 citation statements)

References 51 publications

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“…Recently, AAO structurefunction relationships and mechanistic aspects have been studied in-depth [15][16][17][18][19][20], and its ability to oxidize aromatic (and some aliphatic polyunsaturated) primary alcohols, as well as related aldehydes, has been demonstrated [21,22]. Comparison of the AAO activity for these substrates reveals much lower catalytic efficiency for oxidizing aldehydes than alcohols, due to both lower k cat and higher K m values (Table S1).…”

Section: Discussionmentioning

confidence: 99%

5‐hydroxymethylfurfural conversion by fungal aryl‐alcohol oxidase and unspecific peroxygenase

Ferreira²,

et al. 2015

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Oxidative conversion of 5-hydroxymethylfurfural (HMF) is of biotechnological interest for the production of renewable (lignocellulose-based) platform chemicals, such as 2,5-furandicarboxylic acid (FDCA). To the best of our knowledge, the ability of fungal aryl-alcohol oxidase (AAO) to oxidize HMF is reported here for the first time, resulting in almost complete conversion into 2,5-formylfurancarboxylic acid (FFCA) in a few hours. The reaction starts with alcohol oxidation, yielding 2,5-diformylfuran (DFF), which is rapidly converted into FFCA by carbonyl oxidation, most probably without leaving the enzyme active site. This agrees with the similar catalytic efficiencies of the enzyme with respect to oxidization of HMF and DFF, and its very low activity on 2,5-hydroxymethylfurancarboxylic acid (which was not detected by GC-MS). However, AAO was found to be unable to directly oxidize the carbonyl group in FFCA, and only modest amounts of FDCA are formed from HMF (most probably by chemical oxidation of FFCA by the H 2 O 2 previously generated by AAO). As aldehyde oxidation by AAO proceeds via the corresponding geminal diols (aldehyde hydrates), the various carbonyl oxidation rates may be related to the low degree of hydration of FFCA compared with DFF. The conversion of HMF was completed by introducing a fungal unspecific heme peroxygenase that uses the H 2 O 2 generated by AAO to transform FFCA into FDCA, albeit more slowly than the previous AAO reactions. By adding this peroxygenase when FFCA production by AAO has been completed, transformation of HMF into FDCA may be achieved in a reaction cascade in which O 2 is the only co-substrate required, and water is the only byproduct formed.

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Section: Discussionmentioning

confidence: 99%

5‐hydroxymethylfurfural conversion by fungal aryl‐alcohol oxidase and unspecific peroxygenase

Ferreira²,

et al. 2015

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“…The C-␣ atom of the alcohol moiety contains two hydrogens, of which one will be selectively abstracted. For Pleurotus eryngii AAO (23), it has been shown by using monodeteurated p-methoxybenzyl alcohol that the enzyme abstracts only the R hydrogen, which is closest to the flavin N5, as a hydride. Future structural and kinetic studies will reveal whether HMFO acts via a similar mechanism.…”

Section: Discussionmentioning

confidence: 99%

Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

Dijkman

Fraaije

2014

Appl Environ Microbiol

129

200

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In the search for useful and renewable chemical building blocks, 5-hydroxymethylfurfural (HMF) has emerged as a very promising candidate, as it can be prepared from sugars. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a substitute for petroleum-based terephthalate in polymer production. On the basis of a recently identified bacterial degradation pathway for HMF, candidate genes responsible for selective HMF oxidation have been identified. Heterologous expression of a protein from Methylovorus sp. strain MP688 in Escherichia coli and subsequent enzyme characterization showed that the respective gene indeed encodes an efficient HMF oxidase (HMFO). HMFO is a flavin adenine dinucleotide-containing oxidase and belongs to the glucose-methanol-choline-type flavoprotein oxidase family. Intriguingly, the activity of HMFO is not restricted to HMF, as it is active with a wide range of aromatic primary alcohols and aldehydes. The enzyme was shown to be relatively thermostable and active over a broad pH range. This makes HMFO a promising oxidative biocatalyst that can be used for the production of FDCA from HMF, a reaction involving both alcohol and aldehyde oxidations.

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“…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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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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Stereoselective Hydride Transfer by Aryl‐Alcohol Oxidase, a Member of the GMC Superfamily

Cited by 51 publications

References 51 publications

5‐hydroxymethylfurfural conversion by fungal aryl‐alcohol oxidase and unspecific peroxygenase

5‐hydroxymethylfurfural conversion by fungal aryl‐alcohol oxidase and unspecific peroxygenase

Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

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

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