Crystal Structure of Pyranose 2-Oxidase from the White-Rot Fungus <i>Peniophora</i> sp.<sup>,</sup>

Bannwarth, Michael; Bastian, Sabine; Heckmann-Pohl, Dorothée M.; Giffhorn, Friedrich; Schulz, Georg E.

doi:10.1021/bi048609q

Cited by 50 publications

(58 citation statements)

References 43 publications

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“…Thus, based on the close relationship between the structures of P2⌷x (11,12) and the flavoprotein domain of CD⌯ (33), the reaction mechanism of P2⌷x is expected to involve a hydride-transfer mechanism (32,11). In analogy with CD⌯, which oxidizes the reducing-end glucosyl moiety of cellobiose at C1, ⌯is 548 in P2⌷x (⌯is 689 in CD⌯) is likely to act as a general base to abstract the O2 hydroxyl proton accompanied by transfer of the C2 hydrogen as hydride to the re face of the flavin N5 (11).…”

Section: Discussionmentioning

confidence: 99%

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Structural Basis for Substrate Binding and Regioselective Oxidation of Monosaccharides at C3 by Pyranose 2-Oxidase

Kujawa

Ebner

Leitner

et al. 2006

Journal of Biological Chemistry

235

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Pyranose 2-oxidase (P2Ox) participates in fungal lignin degradation by producing the H 2 O 2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose-and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regioselectivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower k cat , and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Pyranose 2-oxidase (P2Ox, 3 pyranose:oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) is a flavin adenine dinucleotide (FAD)-dependent oxidase present in the hyphal periplasmic space (1) of wood-degrading basidiomycetes (2, 3). These fungi are the only known microorganisms that are capable of fully mineralizing lignin, and P2Ox has a proposed role in the oxidative events (4) of lignin degradation by providing the essential co-substrate, H 2 O 2 , for lignin and manganese peroxidases (5, 6). An alternative hypothesis assigns a role for P2Ox in both H 2 O 2 production and in the reduction of quinones in the periplasm or in the extracellular environment (7). P2Ox from the white-rot fungi Trametes multicolor (Trametes ochracea) and Peniophora gigantea are hitherto the most studied biochemically (7-10) and structurally (11, 12).P2Ox oxidizes a broad range of carbohydrate substrates that are natural constituents of hemicelluloses, allowing most lignocellulose-derived sugars to be utilized. Substrates can be oxidized regioselectively at the C2 position, although some oxidation at C3 can occur as a side reaction (10). For C2 oxidation, D-glucose, D-xylose, and L-sorbose are good or reasonably good substrates, and D-galactose and L-arabinose perform poorly as substrates (7). Based on the catalytic efficiency, k cat /K m , D-glucose (D-Glc) is the best substrate for T. multicolor P2Ox (7). Substrates that are oxidized at C3 were analyzed for P. gigantea P2Ox and include 2-deoxy-D-glucose, 2-keto-D-glucose, and methyl ␤-D-glucosides (13, 10). That oxidation can take place either at C2 or at C3 presupposes two distinct, productive binding modes (referred to here as C2 ox and C3 ox ) for a monosaccharide in the P2Ox active site.P2Ox from T. multicolor is homotetrameric with a molecular mass of 270 kDa (7) where each of the four subunits carries one FAD molecule bound covalently to N ⑀2 (i.e. N3) of His 167 via its 8␣-methyl group (14, 11). The...

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Section: Discussionmentioning

confidence: 99%

“…An alternative hypothesis assigns a role for P2Ox in both H 2 O 2 production and in the reduction of quinones in the periplasm or in the extracellular environment (7). P2Ox from the white-rot fungi Trametes multicolor (Trametes ochracea) and Peniophora gigantea are hitherto the most studied biochemically (7-10) and structurally (11,12).…”

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confidence: 99%

Structural Basis for Substrate Binding and Regioselective Oxidation of Monosaccharides at C3 by Pyranose 2-Oxidase

Kujawa

Ebner

Leitner

et al. 2006

Journal of Biological Chemistry

235

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“…The AAO amino acid sequence revealed moderate homology with glucose oxidase from Aspergillus niger (8), a flavoenzyme in the glucose-methanolcholine oxidases (GMC) oxidoreductase family. The reported molecular model of AAO (9), based on the glucose oxidase crystal structure (10), showed common features with the overall structural topology of bacterial choline oxidase and almond hydroxynitrile lyase (a lyase with oxidoreductase structure), as well as with other members of the GMC family; such as the extracellular flavoenzymes pyranose-2-oxidase and cellobiose dehydrogenase from white-rot basidiomycetes, and bacterial cholesterol oxidase (11)(12)(13)(14)(15). In particular, P. eryngii AAO conserves two histidine residues, His-502 and His-546 (supplemental Fig.…”

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confidence: 94%

Aryl-alcohol Oxidase Involved in Lignin Degradation

Ferreira

Hernández-Ortega

Herguedas

et al. 2009

Journal of Biological Chemistry

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Aryl-alcohol oxidase (AAO) is a FAD-containing enzyme in the GMC (glucose-methanol-choline oxidase) family of oxidoreductases. AAO participates in fungal degradation of lignin, a process of high ecological and biotechnological relevance, by providing the hydrogen peroxide required by ligninolytic peroxidases. In the Pleurotus species, this peroxide is generated in the redox cycling of p-anisaldehyde, an extracellular fungal metabolite. In addition to p-anisyl alcohol, the enzyme also oxidizes other polyunsaturated primary alcohols. Its reaction mechanism was investigated here using p-anisyl alcohol and 2,4-hexadien-1-ol as two AAO model substrates. Steady state kinetic parameters and enzyme-monitored turnover were consistent with a sequential mechanism in which O 2 reacts with reduced AAO before release of the aldehyde product. Pre-steady state analysis revealed that the AAO reductive half-reaction is essentially irreversible and rate limiting during catalysis. Substrate and solvent kinetic isotope effects under steady and presteady state conditions (the latter showing ϳ9-fold slower enzyme reduction when ␣-bideuterated substrates were used, and ϳ13-fold slower reduction when both substrate and solvent effects were simultaneously evaluated) revealed a synchronous mechanism in which hydride transfer from substrate ␣-carbon to FAD and proton abstraction from hydroxyl occur simultaneously. This significantly differs from the general mechanism proposed for other members of the GMC oxidoreductase family that implies hydride transfer from a previously stabilized substrate alkoxide.

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“…The second conserved domain is the C-terminal GMC domain (Pfam 05199). This roughly 150-amino-acid-long domain contains the active-site residues, including a strictly conserved histidine (11).…”

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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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Crystal Structure of Pyranose 2-Oxidase from the White-Rot Fungus Peniophora sp.^,

Cited by 50 publications

References 43 publications

Structural Basis for Substrate Binding and Regioselective Oxidation of Monosaccharides at C3 by Pyranose 2-Oxidase

Structural Basis for Substrate Binding and Regioselective Oxidation of Monosaccharides at C3 by Pyranose 2-Oxidase

Aryl-alcohol Oxidase Involved in Lignin Degradation

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

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Crystal Structure of Pyranose 2-Oxidase from the White-Rot Fungus Peniophora sp.,

Cited by 50 publications

References 43 publications

Structural Basis for Substrate Binding and Regioselective Oxidation of Monosaccharides at C3 by Pyranose 2-Oxidase

Structural Basis for Substrate Binding and Regioselective Oxidation of Monosaccharides at C3 by Pyranose 2-Oxidase

Aryl-alcohol Oxidase Involved in Lignin Degradation

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

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Crystal Structure of Pyranose 2-Oxidase from the White-Rot Fungus Peniophora sp.^,