The Mechanism of Sugar C−H Bond Oxidation by a Flavoprotein Oxidase Occurs by a Hydride Transfer Before Proton Abstraction

Wongnate, Thanyaporn; Surawatanawong, Panida; Chuaboon, Litavadee; Lawan, Narin; Chaiyen, Pimchai

doi:10.1002/chem.201806078

Cited by 12 publications

(7 citation statements)

References 116 publications

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“…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. The reduced flavin is then reoxidized by molecular oxygen yielding hydrogen peroxide as by-product (13).…”

Section: Downloaded Frommentioning

confidence: 99%

Screening and Evaluation of New Hydroxymethylfurfural Oxidases for Furandicarboxylic Acid Production

Viñambres

Espada

Martínez

et al. 2020

Appl Environ Microbiol

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The enzymatic production of 2,5-furandicarboxylic acid (FDCA) from 5-hydroxy-methylfurfural (HMF) has gained interest in recent years, as the renewable precursor of poly(ethylene-2,5-furandicarboxylate) (PEF). 5-Hydroxymethylfurfural oxidases (HMFOs) form a flavoenzyme family with genes annotated in a dozen bacterial species, but only one enzyme purified and characterized to date (after heterologous expression of a Methylovorus sp hmfo gene). This oxidase acts on both furfuryl alcohols and aldehydes being, therefore, able to catalyze the conversion of HMF into FDCA through 2,5-diformylfuran (DFF) and 2,5-formylfurancarboxylic acid (FFCA), with the only need of oxygen as cosubstrate. To enlarge the repertoire of HMFO enzymes available, genetic databases were screened for putative hmfo genes, followed by heterologous expression in Escherichia coli. After unsuccessful trials with other bacterial hmfo genes, HMFOs from two Pseudomonas species were produced as active soluble enzymes, purified and characterized. The Methylovorus sp enzyme was also produced and purified in parallel for comparison. Enzyme stability against temperature, pH and hydrogen peroxide, three key aspects for application, were evaluated (together with optimal conditions for activity) revealing differences between the three HMFOs. Also the kinetic parameters for HMF, DFF and FFCA oxidation were determined, having the new HMFOs higher efficiencies for the oxidation of FFCA, which constitutes the bottleneck in the enzymatic route for FDCA production. These results were used to set up the best conditions for FDCA production by each enzyme, attaining a compromise between optimal activity and half-life under different operation conditions. IMPORTANCE: HMFO is the only enzyme described to date catalyzing by itself the three consecutive oxidation steps to produce FDCA from HMF. Unfortunately, only one HMFO enzyme is currently available for biotechnological application. This availability is enlarged here by the identification, heterologous production, purification and characterization of two new HMFOs from Pseudomonas nitroreducens and an unidentified Pseudomonas species. Compared to the previously known Methylovorus HMFO, the new enzyme from P. nitroreducens exhibits better performance for FDCA production in wider pH and temperature ranges, with higher tolerance to the hydrogen peroxide formed, longer half-life during oxidation, and higher yield and total turnover number in long-term conversions under optimized conditions. All these features are relevant properties for the industrial production of FDCA. In summary, gene screening and heterologous expression can facilitate the selection and improvement of HMFO enzymes as biocatalysts for the enzymatic synthesis of renewable building blocks in the production of bioplastics.

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Section: Downloaded Frommentioning

confidence: 99%

Screening and Evaluation of New Hydroxymethylfurfural Oxidases for Furandicarboxylic Acid Production

Viñambres

Espada

Martínez

et al. 2020

Appl Environ Microbiol

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“…The estimated K D value indicates a high a nity for FAD, comparable to other avoenzymes (for more details, see Supplementary Information) [29][30][31] . The access to the FAD cavity is made through a cavity that contains (i) residues 125 AAHW 128 that is at the same structural position of fungal avinylation motif, 165 STHW 168 in TmP2Ox, that covalently binds FAD, (ii) the substrate loop ( 346 ASPVPLADD 354 ), which in fungal enzymes, is reportedly a dynamic gating segment that ne-tunes the enzymes' reactivity 15,32 , and (iii) the insertion-1 segment (60-93 residues) (Fig. 2d).…”

Section: Resultsmentioning

confidence: 99%

Mechanistic Insights into Glycoside 3-Oxidases Involved in C-Glycoside Metabolism in Soil Microorganisms

Martins

2023

Preprint

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C-glycosides are natural products with important biological activities but are recalcitrant to degradation. Glycoside 3-oxidases (G3Oxs) are newly identified bacterial flavo-oxidases from the glucose-methanol-coline (GMC) superfamily that catalyze the oxidation of C-glycosides with the concomitant reduction of O2 to H2O2. This oxidation is followed by C-C acid/base-assisted bond cleavage in two-step C-deglycosylation pathways. Soil and gut microorganisms have different oxidative enzymes, but the details of their catalytic mechanisms are largely unknown. Here, we report that PsGO3x oxidizes at 50,000-fold higher specificity (kcat/Km) the glucose moiety of mangiferin to 3-keto-mangiferin than free D-glucose to 2-keto-glucose. Analysis of PsG3Ox X-ray crystal structures and PsGO3x in complex with glucose and mangiferin, combined with mutagenesis and molecular dynamics simulations, revealed distinctive features in the topology surrounding the active site that favors catalytically competent conformational states suitable for recognition, stabilization, and oxidation of the glucose moiety of mangiferin. Furthermore, their distinction to pyranose 2-oxidases (P2Oxs) involved in wood decay and recycling is discussed from an evolutionary, structural, and functional viewpoint.

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“…11C). Based on DFT calculations, His548 P2O functions by accepting a proton from the protonated ketone intermediate generated after the hydride transfer step of substrate oxidation in its reductive half‐reaction [55]. In the oxidative half‐reaction, His548 P2O plays a crucial role in C4a‐OOH formation via the PCET mechanism [17].…”

Section: Discussionmentioning

confidence: 99%

Formation and stabilization of C4a‐hydroperoxy‐FAD by the Arg/Asn pair in HadA monooxygenase

Pimviriyakul

Chaiyen

2022

The FEBS Journal

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HadA monooxygenase catalyses the detoxification of halogenated phenols and nitrophenols via dehalogenation and denitration respectively. C4a‐hydroperoxy‐FAD is a key reactive intermediate wherein its formation, protonation and stabilization reflect enzyme efficiency. Herein, transient kinetics, site‐directed mutagenesis and pH‐dependent behaviours of HadA reaction were employed to identify key features stabilizing C4a‐adducts in HadA. The formation of C4a‐hydroperoxy‐FAD is pH independent, whereas its decay and protonation of distal oxygen are associated with pKa values of 8.5 and 8.4 respectively. These values are correlated with product formation within a pH range of 7.6–9.1, indicating the importance of adduct stabilization to enzymatic efficiency. We identified Arg101 as a key residue for reduced FAD (FADH−) binding and C4a‐hydroperoxy‐FAD formation due to the loss of these abilities as well as enzyme activity in HadAR101A and HadAR101Q. Mutations of the neighbouring Asn447 do not affect the rate of C4a‐hydroperoxy‐FAD formation; however, they impair FADH− binding. The disruption of Arg101/Asn447 hydrogen bond networking in HadAN447A increases the pKa value of C4a‐hydroperoxy‐FAD decay to 9.5; however, this pKa was not altered in HadAN447D (pKa of 8.5). Thus, Arg101/Asn447 pair should provide important interactions for FADH− binding and maintain the pKa associated with H2O2 elimination from C4a‐hydroperoxy‐FAD in HadA. In the presence of substrate, the formation of C4a‐hydroxy‐FAD at the hydroxylation step is pH insensitive, and it dehydrates to form the oxidized FAD with pKa of 7.9. This structural feature might help elucidate how the reactive intermediate was stabilized in other flavin‐dependent monooxygenases.

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The Mechanism of Sugar C−H Bond Oxidation by a Flavoprotein Oxidase Occurs by a Hydride Transfer Before Proton Abstraction

Cited by 12 publications

References 116 publications

Screening and Evaluation of New Hydroxymethylfurfural Oxidases for Furandicarboxylic Acid Production

Screening and Evaluation of New Hydroxymethylfurfural Oxidases for Furandicarboxylic Acid Production

Mechanistic Insights into Glycoside 3-Oxidases Involved in C-Glycoside Metabolism in Soil Microorganisms

Formation and stabilization of C4a‐hydroperoxy‐FAD by the Arg/Asn pair in HadA monooxygenase

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