Tuning Chemoenzymatic Pd/Laccase Conformation Toward Optimized Heterogeneous Aerobic Oxidation.

Heterogeneous chemoenzymatic catalysts differing in their spatial organization and relative orientation of their enzymatic laccase and Pd units confined into macrocellular silica foams were tested on veratryl alcohol oxidation. When operating under continuous flow, we show that the catalytic efficiency of hybrids is significantly enhanced when the Pd(II) complex is combined with a laccase exhibiting a surface located lysine next to the T1 oxidation site of the enzyme.

Controlled Immobilization of a Palladium Complex/Laccase Hybrid into a Macrocellular Siliceous Host.

This study investigates the site-directed immobilization of a hybrid catalyst bearing a biquinoline-based-Pd(II) complex (1) and a robust laccase within cavities of a silica foam to favor veratryl alcohol oxidation. We performed the grafting of 1 at a unique surface located lysine of two laccase variants, either at closed (1⊂UNIK ) or opposite position (1⊂UNIK ) of the enzyme oxidation site. After immobilization into the cavities of silica monoliths bearing hierarchical porosity, we show that catalytic activity is dependent on the orientation and loading of each hybrid, 1⊂UNIK being twice as active than 1⊂UNIK (203 TON vs 100 TON) when operating under continuous flow.

Investigation of dirigent like domains from bacterial genomes.

Background: DIRs are mysterious protein that have the ability to scavenge free radicals, which, are highly reactive with molecules in their vicinity. What is even more fascinating is that they carry out from these highly unstable species, a selective reaction (i.e.

Modulation of laccase catalysed oxidations at the surface of magnetic nanoparticles.

We explored the coupling of laccases to magnetic nanoparticles (MNPs) with different surface chemical coating. Two laccase variants offering two opposite and precise orientations of the substrate oxidation site were immobilised onto core-shell MNPs presenting either aliphatic aldehyde, aromatic aldehyde or azide functional groups at the particles surface. Oxidation capabilities of the six-resulting laccase-MNP hybrids were compared on ABTS and coniferyl alcohol.

Tracking light-induced electron transfer toward O in a hybrid photoredox-laccase system.

Photobiocatalysis uses light to perform specific chemical transformations in a selective and efficient way. The intention is to couple a photoredox cycle with an enzyme performing multielectronic catalytic activities. Laccase, a robust multicopper oxidase, can be envisioned to use dioxygen as a clean electron sink when coupled to an oxidation photocatalyst.

Clicked Bifunctional Dendrimeric and Cyclopeptidic Addressable Redox Scaffolds for the Functionalization of Carbon Nanotubes with Redox Molecules and Enzymes.

Carbon nanotube electrodes were modified with ferrocene and laccase using two different click reactions strategies and taking advantage of bifunctional dendrimers and cyclopeptides. Using diazonium functionalization and the efficiency of oxime ligation, the combination of both multiwalled carbon nanotube surfaces and modified dendrimers or cyclopeptides allows the access to a high surface coverage of ferrocene in the order of 50 nmol cm, a 50-fold increase compared to a classic click reaction without oxime ligation of these highly branched macromolecules. Furthermore, this original immobilization strategy allows the immobilization of mono- and bi-functionalized active multicopper enzymes, laccases, copper(I)-catalyzed azide-alkyne cycloaddition.

Coniferyl Alcohol Radical Detection by the Dirigent Protein AtDIR6 Monitored by EPR.

Plant dirigent proteins (DIRs) control the stereoselectivity of the monolignol coniferyl alcohol radical coupling. The main mechanistic hypothesis on this chemo- and stereoselective reaction invokes a binding of coniferyl alcohol radical substrates in the dirigent protein active site so that only one enantiomeric form can be produced. We have studied the influence of the Arabidopsis thaliana AtDIR6 protein on the transient coniferyl alcohol radical by EPR.

Efficiency of Site-Specific Clicked Laccase-Carbon Nanotubes Biocathodes towards O Reduction.

A maximization of a direct electron transfer (DET) between redox enzymes and electrodes can be obtained through the oriented immobilization of enzymes onto an electroactive surface. Here, a strategy for obtaining carbon nanotube (CNTs) based electrodes covalently modified with perfectly control-oriented fungal laccases is presented. Modelizations of the laccase-CNT interaction and of electron conduction pathways serve as a guide in choosing grafting positions.

Production and manipulation of blue copper oxidases for technological applications.

Fungal laccases are robust multicopper oxidoreductases. Perfectly amenable to synthetic evolution, the fungal laccase scaffold is a potential generic for the production of tailored biocatalysts, which, in principle, can be secreted at substantial levels in industrially relevant organisms. In this chapter, the strategy we have developed for the rapid production of hundreds of milligram of laccase variants is detailed.

Mercury Trithiolate Binding (HgS) to a de Novo Designed Cyclic Decapeptide with Three Preoriented Cysteine Side Chains.

Mercury(II) is an unphysiological soft ion with high binding affinity for thiolate ligands. Its toxicity lies in the interactions with low molecular weight thiols including glutathione and cysteine-containing proteins that disrupt the thiol balance and alter vital functions. However, mercury can also be detoxified via interactions with Hg(II)-responsive regulatory proteins such as MerR, which coordinates Hg(II) with three cysteine residues in a trigonal planar fashion (HgS coordination).

Probing the Surface of a Laccase for Clues towards the Design of Chemo-Enzymatic Catalysts.

Systems featuring a multi-copper oxidase associated with transition-metal complexes can be used to perform oxidation reactions in mild conditions. Here, a strategy is presented for achieving a controlled orientation of a ruthenium-polypyridyl graft at the surface of a fungal laccase. Laccase variants are engineered with unique surface-accessible lysine residues.

Visible-Light-Driven Oxidation of Organic Substrates with Dioxygen Mediated by a [Ru(bpy)3 ](2+) /Laccase System.

Oxidation reactions are highly important chemical transformations that still require harsh reaction conditions and stoichiometric amounts of chemical oxidants that are often toxic. To circumvent these issues, olefins oxidation is achieved in mild conditions upon irradiation of an aqueous solution of the complex [Ru(bpy)3 ](2+) and the enzyme laccase. Epoxide formation is coupled to the light-driven reduction of O2 by [Ru(bpy)3 ](2+) /laccase system.

Effect of the Peptidic Scaffold in Copper(II) Coordination and the Redox Properties of Short Histidine-Containing Peptides.

A linear decapeptide containing three His and one Asp residues and a β-turn-inducing dProPro unit was synthesised. A detailed potentiometric, mass spectrometric and spectroscopic study showed that at a 1:1 ratio of CCu /Cpeptide this peptide formed a major [CuH(O(dPro)-Asp)](2+) species (pH range 5.5-7.

Laccases as palladium oxidases.

The first example of a coupled catalytic system involving an enzyme and a palladium(ii) catalyst competent for the aerobic oxidation of alcohol in mild conditions is described. In the absence of dioxygen, the fungal laccase LAC3 is reduced by a palladium(0) species as evidenced by the UV/VIS and ESR spectra of the enzyme. During the oxidation of veratryl alcohol performed in water, at room temperature and atmospheric pressure, LAC3 regenerates the palladium catalyst, is reduced and catalyzes the four-electron reduction of dioxygen into water with no loss of enzyme activity.

Gram-scale production of a basidiomycetous laccase in Aspergillus niger.

We report on the expression in Aspergillus niger of a laccase gene we used to produce variants in Saccharomyces cerevisiae. Grams of recombinant enzyme can be easily obtained. This highlights the potential of combining this generic laccase sequence to the yeast and fungal expression systems for large-scale productions of variants.

Structural and magnetic characterization of a tetranuclear copper(II) cubane stabilized by intramolecular metal cation-π interactions.

A novel tetranuclear copper(II) complex (1) was synthesized from the self-assembly of copper(II) perchlorate and the ligand N-benzyl-1-(2-pyridyl)methaneimine (L(1)). Single-crystal X-ray diffraction studies revealed that complex 1 consists of a Cu4(OH)4 cubane core, where the four copper(II) centers are linked by μ3-hydroxo bridges. Each copper(II) ion is in a distorted square-pyramidal geometry.

Crystallographic snapshots of the reaction of aromatic C-H with O(2) catalysed by a protein-bound iron complex.

Chemical reactions inside single crystals are quite rare because crystallinity is difficult to retain owing to atomic rearrangements. Protein crystals in general have a high solvent content. This allows for some molecular flexibility, which makes it possible to trap reaction intermediates of enzymatic reactions without disrupting the crystal lattice.

The protein environment drives selectivity for sulfide oxidation by an artificial metalloenzyme.

MAGIC Mn-salen mETALLOZYME: The design of an original, artificial, inorganic, complex-protein adduct, has led to a better understanding of the synergistic effects of both partners. The exclusive formation of sulfoxides by the hybrid biocatalyst, as opposed to sulfone in the case of the free inorganic complex, highlights the modulating role of the inorganic-complex-binding site in the protein. Artificial metalloenzymes based on the incorporation of Mn-salen complexes into human serum albumin display high efficiency and selectivity for sulfoxide production during sulfide oxidation.

Selective intracellular accumulation of the major metabolite issued from the activation of the prodrug ethionamide in mycobacteria.

Background: Ethionamide is one of the most widely used drugs for the treatment of multidrug-resistant tuberculosis (MDR-TB). Like isoniazid, and pyrazinamide, ethionamide is a prodrug that needs to be activated by a mycobacterial enzyme. Activation pathways of prodrugs are generally problematic to uncover as they produce intermediates potentially difficult to characterize, to purify and that might prove unstable outside of their cellular context.

Model peptides based on the binding loop of the copper metallochaperone Atx1: selectivity of the consensus sequence MxCxxC for metal ions Hg(II), Cu(I), Cd(II), Pb(II), and Zn(II).

The amino acid sequence MxCxxC is conserved in many soft-metal transporters that are involved in the control of the intracellular concentration of ions such as Cu(I), Hg(II), Zn(II), Cd(II), and Pb(II). A relevant task is thus the selectivity of the motif MxCxxC for these different metal ions. To analyze the coordination properties and the selectivity of this consensus sequence, we have designed two model peptides that mimic the binding loop of the copper chaperone Atx1: the cyclic peptide P(C) c(GMTCSGCSRP) and its linear analogue P(L) (Ac-MTCSGCSRPG-NH2).

Monitoring of the ethionamide pro-drug activation in mycobacteria by (1)H high resolution magic angle spinning NMR.

In this study, we use HRMAS NMR as a non-invasive technique to monitor the in vivo metabolism of a xenobiotic. The antituberculosis Ethionamide is a pro-drug that has to be activated in mycobacteria before inhibiting its cellular target. The use of (1)H HRMAS NMR has allowed to detect a metabolite (ETH*) of the drug directly in living bacteria, even with a spectrometer operating at the relatively low magnetic field of 300MHz.

Solid-phase functionalization of peptides by an alpha-hydrazinoacetyl group.

A novel procedure for the preparation of alpha-hydrazinoacetyl peptides is reported on the basis of the solid-phase coupling of partially or fully Boc-protected hydrazino acetic acid derivatives. The degree of unwanted polymerization of the activated ester during both activation and coupling was found to be significant for the monoprotected derivative BocNHNHCH(2)CO(2)H but could be minimized with the diprotected derivative BocNHNH(Boc)CH(2)CO(2)H and suppressed with the fully protected acid. Despite the instability of the imidocarbonate group toward acids and bases, a low-cost and effective route was sought for the preparation of the tris(Boc)-protected derivative.

HRMAS NMR observation of beta-sheet secondary structure on a water swollen solid support.

In this paper HRMAS NMR was used to investigate whether peptides on a peptidyl resin swollen in aqueous solution can adopt an intramolecular beta-sheet structure. A model peptide YQNPDGSQA, that was previously shown to adopt such a secondary structure in solution, (Blanco et al, J. Am.