Structure and mechanism of galactose oxidase. The free radical site.

Baron, Andrew J.; Stevens, Conrad; Wilmot, Carrie M.; Seneviratne, Kanjula D.; Blakeley, Veronica; Dooley, David M.; Phillips, Simon E. V.; Knowles, Peter F.; McPherson, Michael J.

doi:10.1016/s0021-9258(17)31504-1

Cited by 145 publications

(94 citation statements)

References 29 publications

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“…Unlike other characterized GLOX enzymes, Tr LOx showed weak activity on carbohydrates. These substrates are readily oxidized by the related enzyme, GalOx [ 104 ]. However, Tr LOx was different from AA5_2 enzymes by showing higher activity on monosaccharides, notably xylose and no detectable activity on polysaccharides [ 40 , 105 ].…”

Section: Resultsmentioning

confidence: 99%

A Putative Lignin Copper Oxidase from Trichoderma reesei

Daou

Bisotto

Haon

et al. 2021

JoF

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The ability of Trichoderma reesei, a fungus widely used for the commercial production of hemicellulases and cellulases, to grow and modify technical soda lignin was investigated. By quantifying fungal genomic DNA, T. reesei showed growth and sporulation in solid and liquid cultures containing lignin alone. The analysis of released soluble lignin and residual insoluble lignin was indicative of enzymatic oxidative conversion of phenolic lignin side chains and the modification of lignin structure by cleaving the β-O-4 linkages. The results also showed that polymerization reactions were taking place. A proteomic analysis conducted to investigate secreted proteins at days 3, 7, and 14 of growth revealed the presence of five auxiliary activity (AA) enzymes in the secretome: AA6, AA9, two AA3 enzymes), and the only copper radical oxidase encoded in the genome of T. reesei. This enzyme was heterologously produced and characterized, and its activity on lignin-derived molecules was investigated. Phylogenetic characterization demonstrated that this enzyme belonged to the AA5_1 family, which includes characterized glyoxal oxidases. However, the enzyme displayed overlapping physicochemical and catalytic properties across the AA5 family. The enzyme was remarkably stable at high pH and oxidized both, alcohols and aldehydes with preference to the alcohol group. It was also active on lignin-derived phenolic molecules as well as simple carbohydrates. HPSEC and LC-MS analyses on the reactions of the produced protein on lignin dimers (SS ββ, SS βO4 and GG β5) uncovered the polymerizing activity of this enzyme, which was accordingly named lignin copper oxidase (TrLOx). Polymers of up 10 units were formed by hydroxy group oxidation and radical formation. The activations of lignin molecules by TrLOx along with the co-secretion of this enzyme with reductases and FAD flavoproteins oxidoreductases during growth on lignin suggest a synergistic mechanism for lignin breakdown.

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Section: Resultsmentioning

confidence: 99%

A Putative Lignin Copper Oxidase from Trichoderma reesei

Daou

Bisotto

Haon

et al. 2021

JoF

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“…This may serve as further evidence for the weak intramolecular interaction between the phenoxyl radical moiety and the side chain aromatic ring even in frozen solution. One of the peculiar properties of methylthiophenoxyl radicals is considered to be the preference for the π‐π stacking interaction shown in the active form of GO, [2–8] and the π‐π stacking of the phenoxyl radical moieties has been recently demonstrated as a favorable and effective interaction [19–21,30,50] …”

Section: Discussionmentioning

confidence: 99%

“…Further development of these magnetic materials would be achieved by aligning these magnetic molecules. The Cu II ‐phenoxyl radical species is considered to favor the π‐π stacking interaction based on our recent results, [19–21,30] and as seen in the active form of GO [4–8] . Introduction of such a weak interaction into the phenoxyl radical moiety may lead to the development of the functional magnetic materials.…”

Section: Introductionmentioning

confidence: 96%

“…The phenoxyl radical connects with a sulfur atom from a proximal cysteine through a C−S covalent bond [3] and plays an important role in the catalytic cycle of oxidation of primary alcohol to the corresponding aldehyde [1,2] . This phenoxyl radical moiety is involved in the π‐π stacking interaction with the proximal indole ring for stabilization of the phenoxyl radical [2–8] . In order to understand the detailed properties of Cu II ‐phenoxyl radical complexes, various one‐electron oxidized Cu II ‐phenolate complexes have been synthesized and characterized [9–22] .…”

Section: Introductionmentioning

confidence: 99%

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Solid State Characterization of One‐ and Two‐Electron Oxidized Cu^II‐salen Complexes with para‐Substituents: Geometric Structure‐Magnetic Property Relationship

et al. 2021

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One‐ and two‐electron oxidized CuII complexes of Schiff base ligands with para‐substituted phenolate moieties were synthesized and characterized. The crystal structure of the one‐electron oxidized methylthio‐substituted complex [Cu(MeS‐salen)]SbCl6 revealed the intermolecular π‐π stacking interaction of the two phenolate moieties with those of the neighboring complex cations to form the one‐dimensional chain. Such a π‐π stacking interaction could not be detected in the two‐electron oxidized complex [Cu(MeS‐salen)](SbCl6)2 and the oxidized methoxy‐substituted complexes [Cu(MeO‐salen)](SbF6)n (n=1, 2). Magnetic study of [Cu(MeO‐salen)]SbF6 showed a weak antiferromagnetic interaction between the CuII ion and phenoxyl radical unpaired electron spins, while [Cu(MeS‐salen)]SbCl6 showed a ferromagnetism. Both of the two‐electron oxidized complexes exhibited a relatively strong magnetic interaction between the two radical electrons, while the signs of the electron spins are different.

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“…The active site of galactose oxidase has evolved two distinct but related functions, cofactor assembly and catalysis. The turnover reaction, which has been the focus of the majority of earlier work (2)(3)(4), depends on the presence of a correctly formed redox cofactor in the active site, and a mutational variant lacking the active site Cys 228 that is unable to form the Tyr-Cys cross-link is virtually inactive as a catalyst, even though it binds copper (43). Self-assembly of the Tyr-Cys redox cofactor is a second intrinsic, but noncatalytic, reactivity of the active site (29).…”

Section: Discussionmentioning

confidence: 99%

Cu(I)-dependent Biogenesis of the Galactose Oxidase Redox Cofactor

Whittaker

2003

Journal of Biological Chemistry

112

174

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Galactose oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272. Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen. We have investigated the biogenesis of cofactor-containing galactose oxidase from pregalactose oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II). Mature galactose oxidase forms rapidly following exposure of a pregalactose oxidase-Cu(I) complex to dioxygen (t(1/2) = 3.9s at pH7). In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t(1/2) = 5.1 h). EDTA prevents reaction of pregalactose oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction. The yield of cross-link corresponds to the amount of copper added, although a fraction of the pregalactose oxidase protein is unable to undergo this cross-linking reaction. The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading. The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature galactose oxidase and requires two molecules of O2. Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation. These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism.

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Structure and mechanism of galactose oxidase. The free radical site.

Cited by 145 publications

References 29 publications

A Putative Lignin Copper Oxidase from Trichoderma reesei

A Putative Lignin Copper Oxidase from Trichoderma reesei

Solid State Characterization of One‐ and Two‐Electron Oxidized Cu^II‐salen Complexes with para‐Substituents: Geometric Structure‐Magnetic Property Relationship

Cu(I)-dependent Biogenesis of the Galactose Oxidase Redox Cofactor

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Structure and mechanism of galactose oxidase. The free radical site.

Cited by 145 publications

References 29 publications

A Putative Lignin Copper Oxidase from Trichoderma reesei

A Putative Lignin Copper Oxidase from Trichoderma reesei

Solid State Characterization of One‐ and Two‐Electron Oxidized CuII‐salen Complexes with para‐Substituents: Geometric Structure‐Magnetic Property Relationship

Cu(I)-dependent Biogenesis of the Galactose Oxidase Redox Cofactor

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Solid State Characterization of One‐ and Two‐Electron Oxidized Cu^II‐salen Complexes with para‐Substituents: Geometric Structure‐Magnetic Property Relationship