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The oxidation of benzyl alcohols with the enzyme laccase, under mediation by appropriate mediator compounds, yields carbonylic products, whereas laccase can not oxidise these non-phenolic substrates directly. The oxidation step is performed by the oxidised form of the mediator (Med(ox)), generated on its interaction with laccase. The Med(ox) can follow either an electron transfer (ET) or a radical hydrogen atom transfer (HAT) route of oxidation of the substrates. Experimental evidence is reported that enables unambiguous assessment of the occurrence of either one the oxidation routes with each of the investigated mediators, namely, ABTS, HBT, HPI and VLA. Support to the conclusions is provided by (i) investigating the intermolecular selectivity of oxidation with appropriate substrates, (ii) attempting Hammett correlations for the oxidation of a series of 4-X-substituted benzyl alcohols, (iii) measuring the kinetic isotope effect, (iv) investigating the product pattern with suitable probe precursors. Based on these points, a HAT mechanism results to be followed by the laccase-HBT, laccase-HPI and laccase-VLA systems, whereas an ET route appears feasible in the case of the laccase-ABTS system.

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New mediators of laccase have been comparatively evaluated and ranked towards the benchmark aerobic oxidation of p-MeO-benzyl alcohol. The mechanism of oxidation of this non-phenolic substrate by each mediator, which is initially oxidised by laccase to the Medox form, has been assessed among three alternatives. The latter make the phenoloxidise laccase competent for the indirect oxidation of non-phenolic (and thus ‘unnatural’) substrates. Experimental characterisation of the mediators, by means of spectrophotometric, electrochemical and thermochemical survey, is reported. Clear-cut evidence for the formation of a benzyl radical intermediate in the oxidation of a particular benzyl alcohol with laccase and a {N–OH mediator is attained by means of a trapping experiment. The selectivity of the laccase-catalysed oxidation of two competing lignin and polysaccharide model compounds has been assessed by using the highly proficient 4-MeO-HPI mediator, and found very high in favour of the former model. This evidence is in keeping with the operation of a radical hydrogen-abstraction process that efficiently cleaves the benzylic rather than the aliphatic C–H bond of the two models. Significant is the finding that catechol, i.e., a model of recurring phenolic structures in lignin, once oxidised to aryloxyl radical by laccase is capable to mediate a radical oxidation of non-phenolic compounds. This supports a fully-fledged role of laccase as a delignifying enzyme in nature by way of no other mediators than the very phenolic groups of lignin. Finally, an evaluation of the dissociation energy of the NO–H bond of HBT, which is not accessible experimentally, is provided by the use of a thermochemical cycle and theoretical calculations

Laccases catalyze the one-electron oxidation of a broad range of substrates coupled to the 4 electron reduction of O2 to H2O. Phenols are typical substrates, because their redox potentials (ranging from 0.5 to 1.0 V vs. NHE) are low enough to allow electron abstraction by the T1 Cu(II) that, although a relatively modest oxidant (in the 0.4-0.8 V range), is the electron-acceptor in laccases. The present study comparatively investigated the oxidation performances of Trametes villosa and Myceliophthora thermophila laccases, two enzymes markedly differing in redox potential (0.79 and 0.46 V). The oxidation efficiency and kinetic constants of laccase-catalyzed conversion of putative substrates were determined. Hammett plots related to the oxidation of substituted phenols by the two laccases, in combination with the kinetic isotope effect determination, confirmed a rate-determining electron transfer from the substrate to the enzyme. The efficiency of oxidation was found to increase with the decrease in redox potential of the substrates, and the Marcus reorganisation energy for electron transfer to the T1 copper site was determined. Steric hindrance to substrate docking was inferred because some of the phenols and anilines investigated, despite possessing a redox potential compatible with one-electron abstraction, were scarcely oxidised. A threshold value of steric hindrance of the substrate, allowed for fitting into the active site of T. villosa laccase, was extrapolated from structural information provided by X-ray analysis of T. versicolor lac3B, sharing an identity of 99% at the protein level, thus enabling us to assess the relative contribution of steric and redox properties of a substrate in determining its susceptibility to laccase oxidation. The inferred structural threshold is compatible with the distance between two phenylalanine residues that mark the entrance to the active site. Interaction of the substrate with other residues of the active site is commented on.

GTP cyclohydrolase I catalyzes the conversion of GTP to dihydroneopterin triphosphate. The replacement of histidine 179 by other amino acids affords mutant enzymes that do not catalyze the formation of dihydroneopterin triphosphate. However, some of these mutant proteins catalyze the conversion of GTP to 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5-triphosphate as shown by multinuclear NMR analysis. The equilibrium constant for the reversible conversion of GTP to the ring-opened derivative is approximately 0.1. The wild-type enzyme converts the formylamino pyrimidine derivative to dihydroneopterin triphosphate; the rate is similar to that observed with GTP as substrate. The data support the conclusion that the formylamino pyrimidine derivative is an intermediate in the overall reaction catalyzed by GTP cyclohydrolase I.GTP cyclohydrolase I catalyzes the formation of dihydroneopterin triphosphate from GTP via a mechanistically complex ring expansion. In plants and micro-organisms, the enzyme product serves as the first committed intermediate in the biosynthesis of tetrahydrofolate (1). In animals, the enzyme product is converted to tetrahydrobiopterin, which serves as cofactor for the biosynthesis of catecholamines and of nitric oxide (2-4). Genetic defects of GTP cyclohydrolase I result in severe neurological impairment (5-8).GTP cyclohydrolase I of Escherichia coli is a 247-kDa homodecamer (9, 10). The structure of the protein has been studied by x-ray structure analysis at a resolution of 2.6 Å (11). The torus-shaped protein obeys D 5 symmetry. Each of the 10 equivalent active sites is located at the interface of three adjacent subunits.Brown, Shiota, and their co-workers (12-14) could show the reaction sequence catalyzed by GTP cyclohydrolase I to involve the opening of the imidazole ring of GTP (compound 1, Fig. 1) with release of formate. Carbon atoms 1Ј and 2Ј of the ribose moiety of GTP are then used to form the dihydropyrazine ring of dihydroneopterin triphosphate (compound 5, Fig. 1). However, the mechanistic details of the highly complex enzymecatalyzed reactions are incompletely understood.The catalytic activity of GTP cyclohydrolase is highly sensitive to the replacement of amino acid residues at the active site cavity (11 13 C]formate, phosphoryl chloride, trimethyl phosphate, N,N-dimethylformamide, tri-n-butylamine, and pyrophosphoric acid were purchased from Sigma-Aldrich. All other chemicals were reagent grade.Enzyme Assays-Assay mixtures contained 100 mM Tris hydrochloride, pH 8.5, 100 mM KCl, 2.5 mM EDTA, 1 mM GTP, and protein in a total volume of 450 l. The mixtures were incubated at 37°C, and 100-l aliquots were retrieved at intervals. The formation of dihydroneopterin triphosphate and 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5Ј-phosphate was monitored as follows.Assay of Dihydroneopterin Triphosphate-Aliquots of enzyme assay mixture were mixed with 30 l of a solution containing 1% iodine and 2% KI in 1 M HCl. After incubation for 30 min at ambient temp...

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Aminoxyl radicals (R(2)NO(*)) are a valuable class of reactive intermediates with interesting synthetic and reactivity properties. This Minireview summarizes salient synthetic results obtained in radical oxidations using aminoxyl radicals, and then focuses on reactivity issues arising from recent literature surveys. The structural and reactivity features of the aminoxyl radical and substrate provides a possible explanation of the double reactivity of the aminoxyl radicals. This mechanistic dichotomy between H-atom abstraction and electron-abstraction routes is highlighted in this Minireview.