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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⊂UNIK157) or opposite position (1⊂UNIK71) 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⊂UNIK157 being twice as active than 1⊂UNIK71 (203 TON vs 100 TON) when operating under continuous flow. These systems can be reused 5 times, with an operational activity remaining as high as 40 %. We show that the synergy between 1 and laccase can be tuned within the foam. This work is a proof of concept for controlling the organization of a heterogeneous hybrid catalyst using a Pd/laccase/silica foam. [ABSTRACT FROM AUTHOR]

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., stereoenantioselective) from a well-defined substrate to give a very precise product. Unfortunately, to date, only three products have been demonstrated following studies on DIRs from the plant world, which until now was the kingdom where these proteins had been demonstrated. Within this kingdom, each DIR protein has its own type of substrate. The products identified to date, have on the other hand, a strong economic impact: in agriculture for example, the biosynthesis of (+)-gossypol could be highlighted (a repellent antifood produced by the cotton plant) by the DIRs of cotton. In forsythia plant species, it is the biosynthesis of (−)-pinoresinol, an intermediate leading to the synthesis of podophyllotoxine (a powerful anicancerous agent) which has been revealed. Recently, a clear path of study, potentially with strong impact, appeared by the hypothesis of the potential existence of protein DIR within the genomes of prokaryotes. The possibility of working with this type of organism is an undeniable advantage: since many sequenced genomes are available and the molecular tools are already developed. Even easier to implement and working on microbes, of less complex composition, offers many opportunities for laboratory studies. On the other hand, the diversity of their environment (e.g., soil, aquatic environments, extreme environmental conditions (pH, temperature, pressure) make them very diverse and varied subjects of study. Identifying new DIR proteins from bacteria means identifying new substrate or product molecules from these organisms. It is the promise of going further in understanding the mechanism of action of these proteins and this will most likely have a strong impact in the fields of agricultural, pharmaceutical and/or food chemistry. Results: Our goal is to obtain as much information as possible about these proteins to unlock the secrets of their exceptional functioning. Analyzes of structural and functional genomic data led to the identification of the Pfam PF03018 domain as characteristic of DIR proteins. This domain has been further identified in the sequence of bacterial proteins therefore named as DIR-like (DIRL). We have chosen a multidisciplinary bioinformatic approach centered on bacterial genome identification, gene expression and regulation signals, protein structures, and their molecular information content. The objective of this study was to perform a thorough bioinformatic analysis on these DIRLs to highlight any information leading to the selection of candidate bacteria for further cloning, purification, and characterization of bacterial DIRs. Conclusions: From studies of DIRL genes identification, primary structures, predictions of their secondary and tertiary structures, prediction of DIRL signals sequences, analysis of their gene organization and potential regulation, a list of primary bacterial candidates is proposed. [ABSTRACT FROM AUTHOR]

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. Homogeneous populations of alkyne‐modified laccases are obtained through the reductive amination of a unique surface‐accessible lysine residue selectively engineered near either one or the other of the two copper centers in enzyme variants. Immobilization of the site‐specific alkynated enzymes is achieved by copper‐catalyzed click reaction on azido‐modified CNTs. A highly efficient reduction of O2 at low overpotential and catalytic current densities over −3 mA cm−2 are obtained by minimizing the distance from the electrode surface to the trinuclear cluster. [ABSTRACT FROM AUTHOR]

The amino acid sequence MxCxxC is conserved in many soft-metal transporters that are involved in the control of the intraceIIular concentration of ions such as Cu(l), 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 PC c(GMTCSGCSRP) and its linear analogue pL (Ac-MTCSGCSRPG-NH2). By using complementary analytical and spectroscopic methods, we have demonstrated that 1:1 complexes are obtained with Cu(l) and Hg(II), whereas 1:1 and 1:2 (M:P) species are successively formed with Zn(II), Cd(II), and Pb(II). The complexation properties of the cyclic and linear peptides are very close, but the cyclic compound provides systematicaIIy higher affinity constants than its unstructured analogue. The introduction of a xPGx motif that forms a type II β turn in PC induces a preorganization of the binding loop of the peptide that enhances the stabilities of the complexes (up to 2 orders of magnitude difference for the Hg complexes). The affinity constants were measured in the absence of any reducing agent that would compete with the peptides and range in the order Hg(II) > Cu(l) ⪢ Cd(II) > Pb(II) > Zn(II). This sequence is thus highly selective for Cu(l) compared to the essential ion Zn(II) that could compete in vivo or compared to the toxic ions Cd(II) and Pb(II). Only Hg(II) may be an efficient competitor of Cu(l) for binding to the MxCxxC motif in metaIIoproteins. [ABSTRACT FROM AUTHOR]

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(OdProAsp)]2+ species (pH range 5.5-7.0), in which the Cu2+ ion was bound to the His and Asp residues in square-planar or square-pyramidal geometries. The stability constant corrected for protonated species (log K* [ABSTRACT FROM AUTHOR]

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. A similar approach has not yet been fully implemented in the field of inorganic chemistry. Here, we have combined model chemistry and protein X-ray crystallography to study the intramolecular aromatic dihydroxylation by an arene-containing protein-bound iron complex. The bound complex was able to activate dioxygen in the presence of a reductant, leading to the formation of catechol as the sole product. The structure determination of four of the catalytic cycle intermediates and the end product showed that the hydroxylation reaction implicates an iron peroxo, generated by reductive O2 activation, an intermediate already observed in iron monooxygenases. This strategy also provided unexpected mechanistic details such as the rearrangement of the iron coordination sphere on metal reduction. [ABSTRACT FROM AUTHOR]

Abstract: 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 1H 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. We show that metabolism monitoring of an unlabelled drug at a therapeutically relevant concentration as low as 5μg/ml is within reach of the technique. 1H HRMAS NMR in combination with diffusion filtering leads to the conclusion that the metabolite is located inside the intact cells. The comparison of the metabolite NMR signature with that of synthetic molecules proves the non-identity of ETH* with the ETH derivatives described previously in the literature. [Copyright &y& Elsevier]

A novel procedure for the preparation of α-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[sub 2]CO[sub 2]H but could be minimized with the diprotected derivative BocNHNH(Boc)CH[sub 2]CO[sub 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. The N,N,N'tris(Boc)hydrazinoacetic acid could be introduced on the solid phase after or before peptide elongation using Fmoc/tert-butyl chemistry. In this latter case, HR MAS NMR analysis of model solid supports demonstrated the partial loss of one Boc group during the repetitive piperidine treatments. Despite this slight instability, N,N,N'-tris(Boc)hydrazinoacetic acid was found to be a highly convenient reagent for the robust and easily scalable preparation of hydrazinopeptides in good yield and high purity. [ABSTRACT FROM AUTHOR]

The mono-electronic oxidation of coniferyl alcohol leads to the formation of phenoxy radicals and ultimately to the synthesis of dimeric lignan and neo-lignan products. Coniferyl alcohol and dimeric products are all potential guests for cyclodextrins (CDs) to form noncovalent host–guest inclusion complexes. Herein, the influence of CDs with different cavity volumes (i.e., α, β or CD) on the laccase-driven oxidation of coniferyl alcohol is studied. We clearly show that βCD interacts with the dimeric products and selectively prevents their further oxidation by the enzyme. Moreover, amongst the three dimeric products generated, the system containing a laccase and βCD allows the selective enrichment of pinoresinol; this behaviour is similar to that of plant dirigent proteins. [ABSTRACT FROM AUTHOR]

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. X-ray analysis also evidenced an unusual metal cation-π interaction between the copper ions and phenyl substituents of the ligand. Calculations based on the density functional theory method were used to quantify the strength of this metal-π interaction, which appears as an important stabilizing parameter of the cubane core, possibly acting as a driving parameter in the self-aggregation process. In contrast, using the ligand N-phenethyl-1-(2-pyridyl)methaneimine (L²), which only differs from L¹ by one methylene group, the same synthetic procedure led to a binuclear bis(μ-hydroxo)copper(II) complex (2) displaying intermolecular π-π interactions or, by a slight variation of the experimental conditions, to a mononuclear complex (3). These complexes were studied by X-ray diffraction techniques. The magnetic properties of complexes 1 and 2 are reported and discussed. [ABSTRACT FROM AUTHOR]

Interfacial modulation of laccase activity [Display omitted] • Site-directed immobilization of laccase on core-shell magnetic nanoparticles. • Aliphatic aldehyde, aromatic aldehyde or azide functional groups provide different chemical environment. • Active site orientation at the surface of nanoparticles modulates oxidation of substrates. 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. Herein, we show that the original interfaces created differ substantially in their reactivities with an amplitude from 1 to > 4 folds depending on the nature of the substrate. Taking enzyme orientation into account in the design of surface modification represents a way to introduce selectivity in laccase catalysed reactions. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]