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., 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.

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. Herein, we show that AtDIR6 stabilizes coniferyl alcohol radicals prior to directing their coupling towards the formation of (-)-pinoresinol.

Efficiency of Site-Specific Clicked Laccase-Carbon Nanotubes Biocathodes towards O2 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. 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.

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. It is based on the use of two heterologous expression hosts: the yeast Saccharomyces cerevisiae for a rapid upstream screening and the fungus Aspergillus niger for downstream production. Methods for screening active and nonactive laccase variants, convenient setups for enzyme production in both organisms as well as a methodology for efficient purification of large amounts of recombinant enzymes are given. The general procedure for developing new materials for artificial catalysis is also described.

Enediynes bearing polyfluoroaryl sulfoxide as new antiproliferative agents with dual targeting of microtubules and DNA.

A novel series of enediynes possessing pentafluorophenylsulfoxide have been developed. The innovative compounds possess antiproliferative activity against a broad panel of human cancer cells originating from breast, blood, lung, kidney, colon, prostate, pancreas or skin with IC50 ranging from 0.6 to 3.4 µM. The antiproliferative activity of enediynes in darkness is associated to their ability to compromise microtubule network. In addition, exposure to UV leads to double-stranded DNA cleavage caused by the newly synthesized molecules reducing further their IC50 in nanomolar range against human tumor cells, including chemo-resistant pancreatic cancer cells. Taken together, the examined data demonstrate that enediynes possessing pentafluorosulfoxide are promising molecules in the cancer therapy.

Characterization of Cu(II)-reconstituted ACC Oxidase using experimental and theoretical approaches.

1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a non heme iron(II) containing enzyme that catalyzes the final step of the ethylene biosynthesis in plants. The iron(II) ion is bound in a facial triad composed of two histidines and one aspartate (H177, D179 and H234). Several active site variants were generated to provide alternate binding motifs and the enzymes were reconstituted with copper(II). Continuous wave (cw) and pulsed Electron Paramagnetic Resonance (EPR) spectroscopies as well as Density Functional Theory (DFT) calculations were performed and models for the copper(II) binding sites were deduced. In all investigated enzymes, the copper ion is equatorially coordinated by the two histidine residues (H177 and H234) and probably two water molecules. The copper-containing enzymes are inactive, even when hydrogen peroxide is used in peroxide shunt approach. EPR experiments and DFT calculations were undertaken to investigate substrate's (ACC) binding on the copper ion and the results were used to rationalize the lack of copper-mediated activity.

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. Distinct ruthenium-polypyridyl-modified laccases are obtained by the reductive alkylation of lysine residues precisely located relative to the T1 copper centre of the enzyme. In none of these hybrids does the presence of the graft compromise the catalytic efficiency of the enzyme on the substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Furthermore, the efficiency of the hybrids in olefin oxidation coupled to the light-driven reduction of O2 is highly dependent on the location of the graft at the enzyme surface. Simulated RuII -CuII electron coupling values and distances fit well the observed reactivity and could be used to guide future hybrid designs.

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. The reactivity can be explained by dioxygen acting both as an oxidative agent and as renewable electron acceptor, avoiding the use of a sacrificial electron acceptor.

Characterization of C-terminally engineered laccases.

Extremities of proteins are potent sites for functionalization. Carboxy terminus variants of the Trametes sp. strain C30 LAC3 laccase were generated and produced in Saccharomyces cerevisiae. A variant deleted of the last 13 residues (CΔ) and its 6 His tagged counterpart (CΔ6H) were found active enzymes. The production of CΔ6H resulted in the synthesis of a unusually high proportion of highly glycosylated forms of the enzyme therefore allowing the additional purification of a hyper-glycosylated form of CΔ6H noted CΔ6Hh. Properties of CΔ, CΔ6H and CΔ6Hh were compared. Globally, LAC3 catalytic efficiency was moderately affected by terminal modifications except in CΔ for which the kcat/KM ratio decreased 4 fold (with syringaldazine as substrate) and 10 fold (with ABTS as substrate) respectively. The catalytic parameters kcat and KM of CΔ6H and CΔ6Hh were found to be strictly comparable revealing that over glycosylation does not affect the enzyme catalytic efficiency. To the contrary, in vitro deglycosylation of laccase drastically reduced its activity. So, despite a complex glycosylated pattern observed for some of the variant enzymes, terminal sequences of laccases appear to be appropriate sites for the functionalization/immobilization of laccase.

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.

Species-specificity of the BamA component of the bacterial outer membrane protein-assembly machinery.

The BamA protein is the key component of the Bam complex, the assembly machinery for outer membrane proteins (OMP) in gram-negative bacteria. We previously demonstrated that BamA recognizes its OMP substrates in a species-specific manner in vitro. In this work, we further studied species specificity in vivo by testing the functioning of BamA homologs of the proteobacteria Neisseria meningitidis, Neisseria gonorrhoeae, Bordetella pertussis, Burkholderia mallei, and Escherichia coli in E. coli and in N. meningitidis. We found that no BamA functioned in another species than the authentic one, except for N. gonorrhoeae BamA, which fully complemented a N. meningitidis bamA mutant. E. coli BamA was not assembled into the N. meningitidis outer membrane. In contrast, the N. meningitidis BamA protein was assembled into the outer membrane of E. coli to a significant extent and also associated with BamD, an essential accessory lipoprotein of the Bam complex.Various chimeras comprising swapped N-terminal periplasmic and C-terminal membrane-embedded domains of N. meningitidis and E. coli BamA proteins were also not functional in either host, although some of them were inserted in the OM suggesting that the two domains of BamA need to be compatible in order to function. Furthermore, conformational analysis of chimeric proteins provided evidence for a 16-stranded ß-barrel conformation of the membrane-embedded domain of BamA.

Engineering laccases: in search for novel catalysts.

Laccases (p-diphenol oxidase, EC 1.10.3.2) are blue multicopper oxidases that catalyze the reduction of dioxygen to water, with a concomitant oxidation of small organic substrates. Since the description at the end of the nineteenth century of a factor catalyzing the rapid hardening of the latex of the Japanese lacquer trees (Rhus sp.) exposed to air laccases from different origins (plants, fungi bacteria) have been continuously discovered and extensively studied. Nowadays, molecular evolution and other powerful protein modification techniques offer possibilities to develop tailored laccases for a wide array of applications including drug synthesis, biosensors or biofuel cells. Here, we give an overview on strategies and results of our laboratory in the design of new biocatalysts based on laccases.

Functioning of outer membrane protein assembly factor Omp85 requires a single POTRA domain.

beta-Barrel proteins are present in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. The central component of their assembly machinery is called Omp85 in bacteria. Omp85 is predicted to consist of an integral membrane domain and an amino-terminal periplasmic extension containing five polypeptide-transport-associated (POTRA) domains. We have addressed the function of these domains by creating POTRA domain deletions in Omp85 of Neisseria meningitidis. Four POTRA domains could be deleted with only slight defects in Omp85 function. Only the most carboxy-terminal POTRA domain was essential, as was the membrane domain. Thus, similar to the mitochondrial Omp85 homologue, the functional core of bacterial Omp85 consists of its membrane domain and a single POTRA domain, that is, POTRA5.

Biogenesis of the gram-negative bacterial outer membrane.

The cell envelope of gram-negative bacteria consists of two membranes, the inner and the outer membrane, that are separated by the periplasm. The outer membrane consists of phospholipids, lipopolysaccharides, integral membrane proteins, and lipoproteins. These components are synthesized in the cytoplasm or at the inner leaflet of the inner membrane and have to be transported across the inner membrane and through the periplasm to assemble eventually in the correct membrane. Recent studies in Neisseria meningitidis and Escherichia coli have led to the identification of several machineries implicated in these transport and assembly processes.

Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif.

Integral beta-barrel proteins are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. The assembly of these proteins requires a proteinaceous apparatus of which Omp85 is an evolutionary conserved central component. To study its molecular mechanism, we have produced Omp85 from Escherichia coli in inclusion bodies and refolded it in vitro. The interaction of Omp85 with its substrate proteins was studied in lipid-bilayer experiments, where it formed channels. The properties of these channels were affected upon addition of unfolded outer-membrane proteins (OMPs) or synthetic peptides corresponding to their C-terminal signature sequences. The interaction exhibited species specificity, explaining the inefficient assembly of OMPs from Neisseria in E. coli. Accordingly, the in vivo assembly of the neisserial porin PorA into the E. coli outer membrane was accomplished after adapting its signature sequence. These results demonstrate that the Omp85 assembly machinery recognizes OMPs by virtue of their C-terminal signature sequence.

Subcomplexes from the Xcp secretion system of Pseudomonas aeruginosa.

In Gram-negative bacteria, most of the sec-dependent exoproteins are secreted via the type II secretion system (T2SS or secreton). In Pseudomonas aeruginosa, T2SS consists of 12 Xcp proteins (XcpA and XcpP to XcpZ) organized as a multiproteic complex within the envelope. In this study, by a co-purification approach using a His-tagged XcpZ as a bait, XcpY and XcpZ were found associated together to constitute the most stable functional unit so far isolated from the P. aeruginosa secreton. This subcomplex was also found to interact with XcpR and XcpS to form a XcpRSYZ complex which was isolated under native conditions. Another component, XcpP was not found to be associated to the complex but the results suggest that it can transiently interact with the XcpYZ subcomplex in vivo.

Role of XcpP in the functionality of the Pseudomonas aeruginosa secreton.

In gram-negative bacteria, most signal-peptide-dependent exoproteins are secreted via the type II secretion system (T2SS or secreton). In Pseudomonas aeruginosa, T2SS consists of twelve Xcp proteins (XcpA and XcpP to XcpZ) thought to be organized as a multiproteic complex within the envelope. Although well conserved, T2SS are known to be species-specific, namely for distant organisms, and this characteristic was thought to involve XcpP. To check which domain of XcpP could be involved in the species specificity, hybrid proteins were generated using protein domain swapping between P. aeruginosa XcpP and homolog proteins of either Erwinia chrysanthemi or Pseudomonas alcaligenes. The results obtained with hybrid proteins constructed by exchanging the C-terminal domains of P. aeruginosa and E. chrysanthemi suggested that XcpP interacts with XcpQ, probably via its C-terminal domain. More interestingly, the data obtained with a hybrid protein containing the C-terminal part of the P. alcaligenes XcpP homolog, showed that the wild-type C-terminal end plays a very important role in the function of the protein and is required both for a correct interaction with XcpQ and for modulating the opening of the secreton channel.

Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus.

Gram-negative bacteria have evolved several types of secretion mechanisms to release proteins into the extracellular medium. One such mechanism, the type II secretory system, is a widely conserved two-step process. The first step is the translocation of signal peptide-bearing exoproteins across the inner membrane. The second step, the translocation across the outer membrane, involves the type II secretory apparatus or secreton. The secretons are made up of 12-15 proteins (Gsp) depending on the organism. Even though the systems are conserved, heterologous secretion is mostly species restricted. Moreover, components of the secreton are not systematically exchangeable, especially with distantly related microorganisms. In closely related species, two components, the GspC and GspD (secretin) family members, confer specificity for substrate recognition and/or secreton assembly. We used Pseudomonas aeruginosa as a model organism to determine which domains of XcpP (GspC member) are involved in specificity. By constructing hybrids between XcpP and OutC, the Erwinia chrysanthemi homologue, we identified a region of 35 residues that was not exchangeable. We showed that this region might influence the stability of the XcpYZ secreton subcomplex. Remarkably, XcpP and OutC have domains, coiled-coil and PDZ, respectively, which exhibit the same function but that are structurally different. Those two domains are exchangeable and we provided evidence that they are involved in the formation of homomultimeric complexes of XcpP.

Identification of XcpZ domains required for assembly of the secreton of Pseudomonas aeruginosa.

Most of the exoproteins secreted by Pseudomonas aeruginosa are transported via the type II secretion system. This machinery, which is widely conserved in gram-negative bacteria, consists of 12 Xcp proteins organized as a multiprotein complex, also called the secreton. We previously reported that the mutual stabilization of XcpZ and XcpY plays an important role in the assembly of the secreton. In this study, we engineered variant XcpZ proteins by using linker insertion mutagenesis. We identified three distinct regions of XcpZ required for both the stabilization of XcpY and the functionality of the secreton. Interestingly, we also demonstrated that another component of the machinery, XcpP, can modulate the stabilizing activity of XcpZ on XcpY.