Giuliano Sciara

French National Institute for Agriculture, Food, and Environment (INRAE) | INRAE · Biodiversité et Biotechnologie Fongiques (BBF)

Antibiotics play a crucial role in human and animal medical healthcare, but widespread use and overuse of antibiotics poses alarming health and environmental issues. Fluoroquinolones constitute a class of antibiotics that has already become ubiquitous in the environment, and their increasing use and high persistence prompt growing concern. Here we...

Coriolopsis gallica (Cga) is a white-rot fungus renowned for its ability to secrete ligninolytic enzymes that are capable of oxidizing phenolic compounds. This study aimed to investigate the biochemical characteristics of a dye-decolorizing peroxidase named CgaDyP1 and test its ability to biotransform antibiotics. CgaDyP1 was cloned and heterologou...

Continued widespread use of antibiotics, especially fluoroquinolones, raises environmental concerns, as its driving bacterial resistance and disrupts microbial ecosystems. Here we investigate the biodegradation of ten fluoroquinolone antibiotics (six for medical use and four for veterinary use) by ligninolytic fungi, including Trametes versicolor,...

Despite various plans to rationalize antibiotic use, antibiotic resistance in environmental bacteria is increasing due to the accumulation of antibiotic residues in the environment. This study aimed to test the ability of basidiomycete fungal strains to biotransform the antibiotic levofloxacin, a widely-used third-generation broad-spectrum fluoroqu...

We provide the first crystallographic structure of a green mononuclear monodomain cupredoxin. Analysis of the structure suggests that the coupled distortion model might not explain the behaviour of some cupredoxins.

Here, we report work on developing an enzymatic process to improve the functionalities of industrial lignin. A kraft lignin sample prepared from marine pine was treated with the high-redox-potential laccase from the basidiomycete fungus Pycnoporus cinnabarinus at three different concentrations and pH conditions, and with and without the chemical me...

Lignins are by-products from biorefinery and paper industries. Their chemical structures are complex, and they are currently mainly used for energy and heat production generated by their combustion. In a context of higher value applications of lignins, lignin-based composites with lignosulfonate matrix and wood fibers reinforcement were pre-treated...

Filamentous fungi play a central regulatory role on Earth, notably in the global carbon cycle. Regardless of their lifestyle, filamentous fungi need to remodel their own cell wall (mostly composed of polysaccharides) to grow and proliferate.

Filamentous fungi are keystone microorganisms in the regulation of many processes occurring on Earth, such as plant biomass decay, pathogenesis as well as symbiotic associations. In many of these processes, fungi secrete carbohydrate-active enzymes (CAZymes) to modify and/or degrade carbohydrates. Ten years ago, while evaluating the potential of a...

The wastewater from hospitals, pharmaceutical industries and more generally human and animal dejections leads to environmental releases of antibiotics that cause severe problems for all living organisms. The aim of this study was to investigate the capacity of three fungal strains to biotransform the fluoroquinolone levofloxacin. The degradation pr...

The textile industry generates huge volumes of colored wastewater that require multiple treatments to remove persistent toxic and carcinogenic dyes. Here we studied the decolorization of a recalcitrant azo dye, Reactive Black 5, using laccase-like active cell-free supernatant from Coriolopsis gallica. Decolorization was optimized in a 1 mL reaction...

Laccase-treated wood fibres were tested for small-scale wet-process manufacture of hardboards. Two laccases of distinct redox potentials, one from Pycnoporus cinnabarinus and one from Myceliophthora thermophila, were compared in terms of their effect on the physical–chemical properties of the treated fibres and hardboards. Wood fibres were produced...

Background Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenz...

Mangrove sediments from New Caledonia were screened for xylanase sequences. One enzyme was selected and characterized both biochemically and for its industrial potential. Using a specific cDNA amplification method coupled with a MiSeq sequencing approach, the diversity of expressed genes encoding GH11 xylanases was investigated beneath Avicenia mar...

The functional diversity of the New Caledonian mangrove sediments was examined, observing the distribution of fungal dye-decolorizing peroxidases (DyPs), together with the complete biochemical characterization of the main DyP. Using a functional metabarcoding approach, the diversity of expressed genes encoding fungal DyPs was investigated in surfac...

Background: Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoen...

Only a few studies have examined how marine-derived fungi and their enzymes adapt to salinity and plant biomass degradation. This work concerns the production and characterisation of an oxidative enzyme identified from the transcriptome of marine-derived fungus Stemphylium lucomagnoense. The laccase-encoding gene SlLac2 from S. lucomagnoense was cl...

Even if the ocean represents a large part of Earth’s surface, only a few studies describe marine-derived fungi compared to their terrestrial homologues. In this ecosystem, marine-derived fungi have had to adapt to the salinity and to the plant biomass composition. This articles studies the growth of five marine isolates and the tuning of lignocellu...

Background: Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost-effective processes amenable to biotechnological app...

Background : Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost–effective processes amenable to biotechnological app...

Background Environmental pollution is one of the major problems that world is facing to date. Several approaches are been studied and oxidative enzymes from microbial organisms represent an eco-friendly and cost–effective processes, amenable to biotechnological applications, as for instance industrial dye decolorization. The aim of this study was t...

Two laccase-encoding genes from the marine-derived fungus Pestalotiopsis sp. have been cloned in Aspergillus niger for heterologous production, and the recombinant enzymes have been characterized to study their physicochemical properties, their ability to decolorize textile dyes for potential biotechnological applications, and their activity in the...

Mononuclear cupredoxins contain a type 1 copper center with a trigonal or tetragonal geometry usually maintained by four ligands, a cystein, two histidines and a methionine. The recent discovery of new members of this family with unusual properties demonstrates, however, the versatility of this class of proteins. Changes in their ligand set lead to...

Background: OleP is a cyt P450 from S. antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism. Methods: The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s u...

Cupredoxins are widespread copper-binding proteins, mainly involved in electron transfer pathways. They display a typical rigid greek key motif consisting of an eight stranded β-sandwich. A fascinating feature of cupredoxins is the natural diversity of their copper center geometry. These geometry variations give rise to drastic changes in their col...

The CDP-alcohol phosphotransferase (CDP-AP) family of integral membrane enzymes catalyses the transfer of a substituted phosphate group from a CDP-linked donor to an alcohol acceptor. This is an essential reaction for phospholipid biosynthesis across all kingdoms of life, and it is catalysed solely by CDP-APs. Here we report the 2.0 Å resolution cr...

After decades of absent or lackluster growth, recent years have at long last witnessed an exponential growth in the number of novel membrane protein structures determined. Every single achievement has had a tremendous impact on the scientific community, providing an unprecedented wealth of information that typically only an atomic resolution struct...

Siphoviridae is the most abundant viral family on earth which infects bacteria as well as archaea. All known siphophages infecting gram+ Lactococcus lactis possess a baseplate at the tip of their tail involved in host recognition and attachment. Here, we report analysis of the p2 phage baseplate structure by X-ray crystallography and electron micro...

We report here the characterization of several large structural protein complexes forming the baseplates (or part of them) of Siphoviridae phages infecting Lactococcus lactis: TP901-1, Tuc2009 and p2. We revisited a "block cloning" expression strategy and extended this approach to genomic fragments encoding proteins whose interacting partners have...

G protein-coupled receptors (GPCRs) represent approximately 3% of the human proteome. They are involved in a large number of diverse processes and, therefore, are the most prominent class of pharmacological targets. Besides rhodopsin, X-ray structures of classical GPCRs have only recently been resolved, including the beta1 and beta2 adrenergic rece...

Our aim is to shed light on the conservation of potential ligand docking sites that play an important role in ligand dynamics of globins by using the technique of filling internal cavities naturally present in hemoglobin and myoglobin with xenon atoms. In particular, we present the high resolution structures of the Xe‐adduct of deoxygenated wild ty...

Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the t...

We characterize a new class of crystallizing agent for soluble protein crystallization compatible with both pharmaceutical processes and high-resolution structure determination in biocrystallography. Poloxamers are amphiphilic nonionic multiblock polymers used in the cosmetic and pharmaceutical industries. Poloxamer P188 (EO75PO31EO75) is generally...

Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the t...

We report here a general strategy to overproduce and characterize membrane transporters. To illustrate our approach, we selected one member of the CorA transporter family among four tested that belonged to different species. This approach is transposable to other membrane proteins and involves the following steps: (i) cloning by homologous recombin...

We report here the characterization of the nonstructural protein ORF12 of the virulent lactococcal phage p2, which belongs to the Siphoviridae family. ORF12 was produced as a soluble protein, which forms large oligomers (6- to 15-mers) in solution. Using anti-ORF12 antibodies, we have confirmed that ORF12 is not found in the virion structure but is...

In the course of a crystallographic study of the Methanosarcina mazei CorA transporter, the membrane protein was obtained with at least 95% purity and was submitted to crystallization trials. Small crystals (<100 microm) were grown that diffracted to 3.42 A resolution and belonged to space group R32, with unit-cell parameters a = b = 145.74, c = 51...

Phages infecting Lactococcus lactis, a Gram-positive bacterium, are a recurrent problem in the dairy industry. Despite their economical importance, the knowledge on these phages, belonging mostly to Siphoviridae, lags behind that accumulated for members of Myoviridae. The three-dimensional structures of the receptor-binding proteins (RBP) of three...

Erythromycin A is produced by Saccharopolyspora erythraea via a secondary metabolic pathway using several steps including glycosylations and hydroxylations of the first macrolide intermediate 6-deoxyerythronolide B. Erythromycin C-12 hydroxylase (CYP113A1), the P450 cytochrome active in the final stages of erythromycin biosynthesis, was cloned and...

Work carried out over the last 30 years unveiled the role of structural dynamics in controlling protein function. Cavity networks modulate structural dynamics trajectories and are functionally relevant; in globins they have been assigned a role in ligand migration and docking. These findings raised renewed interest for time-resolved structural inve...

Although conformational changes are essential for the function of proteins, little is known about their structural dynamics at atomic level resolution. Myoglobin (Mb) is the paradigm to investigate conformational dynamics because it is a simple globular heme protein displaying a photosensitivity of the iron-ligand bond. Upon laser photodissociation...

Crystals of the Met derivative of the sperm whale myoglobin triple mutant Mb-YQR [L(B10)Y, H(E7)Q and T(E10)R] were grown under microgravity conditions and on earth by vapour diffusion. A comparison of crystal quality after complete data collection and processing shows how microgravity-grown crystals diffract to better resolution and lead to consid...

ActVA-Orf6 monooxygenase from Streptomyces coelicolor that catalyses the oxidation of an aromatic intermediate of the actinorhodin biosynthetic pathway is a member of a class of small monooxygenases that carry out oxygenation without the assistance of any of the prosthetic groups, metal ions or cofactors normally associated with activation of molec...

The solution structure of oxidized cytochrome c553(71 amino acid residues) from the Gram-positive bacterium Bacillus pasteurii is here reported and compared with the available crystal structure. The solution structure is obtained from 1609 meaningful NOE data (22.7 per residue), 76 dihedral angles, and 59 pseudocontact shifts. The root mean square...

The solution structure of oxidized cytochrome c(553) (71 amino acid residues) from the Gram-positive bacterium Bacillus pasteurii is here reported and compared with the available crystal structure. The solution structure is obtained from 1609 meaningful NOE data (22.7 per residue), 76 dihedral angles, and 59 pseudocontact shifts. The root mean squa...