The Oxidation of Thiols by Flavoprotein Oxidases: a Biocatalytic Route to Reactive Thiocarbonyls

Abstract

Flavoprotein oxidases are a diverse class of biocatalysts, most of which catalyze the oxidation of CÀO, CÀN, or CÀC bonds. Flavoprotein oxidases that are known to catalyze the oxidation of CÀS bonds are rare, being limited to enzymes that catalyze the oxidative cleavage of thioethers. Herein, we report that various flavoprotein oxidases, previously thought to solely act on alcohols, also catalyze the oxidation of thiols to thiocarbonyls. These results highlight the versatility of enzymatic catalysis and provide a potential biocatalytic route to reactive thiocarbonyl compounds, which have a variety of applications in synthetic organic chemistry.Flavoprotein oxidases constitute a diverse class of oxidoreductases, which contain a flavin cofactor in the form of flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). They catalyze the oxidation of a wide range of substrates, with molecular oxygen functioning as the final oxidant. Their catalytic cycle consists of two half reactions. During the reductive half reaction two electrons are transferred from the substrate to the flavin cofactor, a process which is generally believed to occur through the direct transfer of a hydride anion to the N5 atom of the flavin. [1,2] In the oxidative half reaction the flavin cofactor is reoxidized by molecular oxygen, which is reduced to hydrogen peroxide. Unlike many other oxidative enzymes, for example dehydrogenases, oxidases do not require stoichiometric amounts of expensive organic cofactors, making them interesting candidates for biocatalytic exploitation. Most of the characterized flavoprotein oxidases catalyze the oxidation of CÀO, CÀN, or CÀC bonds to the corresponding double bonds.[3] A small number of flavoprotein oxidases are known to oxidize sulfurcontaining compounds. Prenylcysteine lyase (Enzyme Commission (EC) number 1.8.3.5) and farnesylcysteine lyase (EC 1.8.3.6) catalyze the oxidative cleavage of thioethers into aldehydes and thiols, whereas sulfhydryl oxidases (EC 1.8.3.2 and 1.8.3.3) catalyze the formation of disulfide bonds from thiols. So far, no flavoprotein oxidases have been identified that catalyze the oxidation of thiols to thiocarbonyl compounds. Herein, we present the finding that a number of flavin-dependent oxidases, that hitherto had been thought to act solely on alcohols, also oxidize thiols that are structurally similar to their alcohol substrates, yielding the corresponding thiocarbonyls.Alditol oxidase from Streptomyces coelicolor (AldO; EC 1.1.3.41) is a flavin-dependent oxidase that catalyzes the regioselective oxidation of the primary hydroxy groups of a broad range of polyols, with the best substrates being alditols, such as xylitol, d-sorbitol, and l-threitol.[4, 5] AldO displays stereoselectivity: for example, d-sorbitol is converted into its oxidized form significantly more efficiently than its C2 epimer d-mannitol. AldO belongs to the vanillyl alcohol oxidase (VAO) family of structurally homologous flavoproteins. These flavoproteins contain a conserved FAD-binding domain ...

Screening and Evaluation of New Hydroxymethylfurfural Oxidases for Furandicarboxylic Acid Production

“…Methylovorus sp strain M688 (MetspHMFO), a homologue of the enzyme encoded by the hmfH gene of C. basilensis, that was produced in Escherichia coli (6). This flavoenzyme, with a FAD molecule as prosthetic group, is active on primary alcohols, primary thiols and hydrated aldehydes (6,7). Its catalytic mechanism, similar to that of other GMC oxidases (8)(9)(10)(11), involves a proton transfer from the hydroxyl (or thiol) group to a conserved catalytic base, MetspHMFO His467 (12), and the hydride abstraction from the substrate α-carbon by the oxidized flavin.…”

Techniques for Enzyme Purification

“…We used the His-tag technology for the purification of several oxidoreductases including galactonolactone dehydrogenase from Arabidopsis thaliana [72], styrene monooxygenase from R. opacus 1CP [73,74], 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1 and Pseudomonas alcaligenes [26,75], ene reductase from R. opacus CP1 [76], pyranose 2-oxidase from Arthrobacter siccitolerans [77], styrene monooxygenase reductase from R. opacus 1CP [78], vanillyl alcohol oxidase from P. simplicissimum [79], eugenol oxidase from Rhodococcus jostii RHA1 [80], 5-(hydroxymethyl)furfural oxidase from Methylovorus sp. strain MP688 [81,82], and 4-hydroxybenzoate hydroxylase from Cupriavidus necator [83].…”

“…AldO accepts various polyols, with xylitol and sorbitol being the preferred substrates. Intriguingly, AldO also oxidizes thiols such as DTT to the corresponding thiocarbonyls [82].…”

Positive Impact of Natural Deep Eutectic Solvents on the Biocatalytic Performance of 5-Hydroxymethyl-Furfural Oxidase

“…5-Hydroxymethylfurfural oxidase (HMFO, EC 1.1.3.47) from Methylovorus sp. strain MP688 is a flavin-containing oxidase that only requires molecular oxygen as a mild oxidant to catalyze various oxidation reactions which include selective oxidations of alcohols, aldehydes and even thiols [1][2][3]. Uniquely, this oxidative biocatalyst is capable of performing the three-step oxidation of 5-hydroxymethylfurfural (HMF), to the valuable furan-2,5-dicarboxylic acid (FDCA) [4], a chemical platform for the production of biobased polymers such as poly(ethylene-furandicarboxylate) (PEF).…”