Vincent Monchois

Characterization of Leuconostoc mesenteroides NRRL B-512F dextransucrase (DSRS) and identification of amino-acid residues playing a key role in enzyme activity

Dextransucrase (DSRS) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes the synthesis of soluble dextran from sucrose or oligosaccharides when acceptor molecules, like maltose, are present. The L. mesenteroides NRRL B-512F dextransucrase-encoding gene (dsrS) was amplified by the polymerase chain reaction and cloned in an overexpression plasmid. The characteristics of DSRS were found to be similar to the characteristics of the extracellular dextransucrase produced by L. mesenteroides NRRL B-512F. The enzyme also exhibited a high homology with other glucosyltransferases. In order to identify critical amino acid residues, the DSRS sequence was aligned with glucosyltransferase sequences and four amino acid residues were selected for site- directed mutagenesis experiments: aspartic acid 511, aspartic acid 513, aspartic acid 551 and histidine 661. Asp-511, Asp-513 and Asp-551 were independently replaced with asparagine and His-661 with arginine. Mutation at Asp-511 and Asp-551 completely suppressed dextran and oligosaccharide synthesis activities, showing that at least two carboxyl groups (Asp-511 and Asp-551) are essential for the catalysis process. However, glucan-binding properties were retained, showing that DSRS has a two-domain structure like other glucosyltransferases. Mutations at Asp-513 and His-661 resulted in greatly reduced dextransucrase activity. According to amino acid sequence alignments of glucosyltransferases, α-amylases or cyclodextrin glucanotransferases, His-661 may have a hydrogen-bonding function.

Escherichia coli ykfE ORFan Gene Encodes a Potent Inhibitor of C-type Lysozyme

The complete nucleotide sequences of over 37 microbial and three eukaryote genomes are already publicly available, and more sequencing is in progress. Despite this accumulation of data, newly sequenced microbial genomes continue to reveal up to 50% of functionally uncharacterized “anonymous” genes. A majority of these anonymous proteins have homologues in other organisms, whereas the rest exhibit no clear similarity to any other sequence in the data bases. This set of unique, apparently species-specific, sequences are referred to as ORFans. The biochemical and structural analysis of ORFan gene products is of both evolutionary and functional interest. Here we report the cloning and expression ofEscherichia coli ORFan ykfE gene and the functional characterization of the encoded protein. Under physiological conditions, the protein is a homodimer with a strong affinity for C-type lysozyme, as revealed by co-purification and co-crystallization. Activity measurements and fluorescence studies demonstrated that the YkfE gene product is a potent C-type lysozyme inhibitor (K i ≈ 1 nm). To denote this newly assigned function, ykfE has now been registered under the new gene name Ivy (inhibitor ofvertebrate lysozyme) at the E. coligenetic stock center.

Cloning and sequencing of a gene coding for a novel dextransucrase from Leuconostoc mesenteroides NRRL B-1299 synthesizing only α(1–6) and α(1–3) linkages

Abstract The coding region for a Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene ( dsrA ) was isolated and sequenced. Using a pair of primers designed on the basis of two highly conserved amino-acid (aa) sequences in L. mesenteroides NRRL B-512F dextransucrase and streptococcal glucosyltransferases (GTFs), a fragment of dsrA was amplified by the polymerase chain reaction (PCR). This PCR product was used as an hybridization probe to isolate a 1.8-kb fragment identified as the central region of dsrA . Cleavage by Sac I of this fragment allowed two probes to be obtained to isolate the 5′ and the 3′ ends of dsrA . The nucleotide sequence of the dsrA gene was determined and found to consist of an open reading frame (ORF) of 4870 base pairs (bp) coding for a 1290-aa protein with an M r of 145 590. The aa sequence exhibited a high similarity with other GTFs. The two domains previously described in GTFs are conserved in DSRA: an N-terminal conserved domain and a C-terminal domain composed of a series of repeats. Surprisingly, the expected signal peptide was not detected. The entire gene was reconstructed and the activity of DSRA was investigated. The dextran produced appeared to be composed of 85% α(1–6) and 15% α(1–3) linkages and the oligosaccharides synthesized in the presence of maltose were mainly composed of α(1–6) linkages. This enzyme is a novel dextransucrase from L. mesenteroides NRRL B-1299 producing no α(1–2) linkages and is the first glucosyltransferase having no signal peptide described.

Structure and Evolution of the Ivy Protein Family, Unexpected Lysozyme Inhibitors in Gram-Negative Bacteria.

Part of an ancestral bactericidal system, vertebrate C-type lysozyme targets the peptidoglycan moiety of bacterial cell walls. We report the crystal structure of a protein inhibitor of C-type lysozyme, the Escherichia coli Ivy protein, alone and in complex with hen egg white lysozyme. Ivy exhibits a novel fold in which a protruding five-residue loop appears essential to its inhibitory effect. This feature guided the identification of Ivy orthologues in other Gram-negative bacteria. The structure of the evolutionary distant Pseudomonas aeruginosa Ivy orthologue was also determined in complex with hen egg white lysozyme, and its antilysozyme activity was confirmed. Ivy expression protects porous cell-wall E. coli mutants from the lytic effect of lysozyme, suggesting that it is a response against the permeabilizing effects of the innate vertebrate immune system. As such, Ivy acts as a virulence factor for a number of Gram-negative bacteria-infecting vertebrates.

Crystal structure of Escherichia coli DkgA, a broad-specificity aldo-keto reductase.

The Structural and Genomics Informa-tion Laboratory is involved in a Structural and FunctionalGenomics program (BIGS; http://www.igs.cnrs-mrs.fr/str_gen/) aiming at the discovery of new antibacterialtargets among proteins that are ubiquitous in bacterialpathogens, exhibiting good sequence conservation, butwhose precise biochemical or cellular functions remainunknown.Comprehensivebioinformaticsandcomparativegenomics analyses were performed according to thesecriteria, resulting in the selection of 110

Structural characterization of CA1462, the Candida albicans thiamine pyrophosphokinase.

BackgroundIn search of new antifungal targets of potential interest for pharmaceutical companies, we initiated a comparative genomics study to identify the most promising protein-coding genes in fungal genomes. One criterion was the protein sequence conservation between reference pathogenic genomes. A second criterion was that the corresponding gene in Saccharomyces cerevisiae should be essential. Since thiamine pyrophosphate is an essential product involved in a variety of metabolic pathways, proteins responsible for its production satisfied these two criteria.ResultsWe report the enzymatic characterization and the crystallographic structure of the Candida albicans Thiamine pyrophosphokinase. The protein was co-crystallized with thiamine or thiamine-PNP.ConclusionThe presence of an inorganic phosphate in the crystallographic structure opposite the known AMP binding site relative to the thiamine moiety suggests that a second AMP molecule could be accommodated in the C. albicans structure. Together with the crystallographic structures of the enzyme/substrate complexes this suggests the existence of a secondary, less specific, nucleotide binding site in the Candida albicans thiamine pyrophosphokinase which could transiently serve during the release or the binding of ATP. The structures also highlight a conserved Glutamine residue (Q138) which could interact with the ATP α-phosphate and act as gatekeeper. Finally, the TPK/Thiamine-PNP complex is consistent with a one step mechanism of pyrophosphorylation.

Proficient Target Selection in Structural Genomics by In Vitro Protein Expression on Gateway Recombination Plasmids

The numerous whole genome-sequencing projects of the recent genomic era resulted in identifying a huge number of genes with unknown functions, which are now waiting to be characterized. The bioinformatic annotation of prokaryotic genomes, for instance, identifies most of the genes, but leaves 30%–50% of them without any functional attributes [1]. To face this new challenge, many “functional genomics” and “structural genomics” initiatives have been launched throughout the world. One of the goals of structural genomics is to accelerate the discovery of original protein folds as the basis to better understand the protein folding mechanisms, and, thus, improve the performances of computer programs for ab initio 3-D structure modeling or “threading” of proteins. The 3-D structure determination of proteins of known function usually provides detailed insights into their mechanisms of action at the molecular level. It is now expected that the 3-D structure of proteins of unknown function will, in many cases, reveal their similarity to previously described protein families and will immediately provide functional hints that can then be tested experimentally.

The conserved Candida albicans CA3427 gene product defines a new family of proteins exhibiting the generic periplasmic binding protein structural fold.

Nosocomial diseases due to Candida albicans infections are in constant rise in hospitals, where they cause serious complications to already fragile intensive care patients. Antifungal drug resistance is fast becoming a serious issue due to the emergence of strains resistant to currently available antifungal agents. Thus the urgency to identify new potential protein targets, the function and structure of which may guide the development of new antifungal drugs. In this context, we initiated a comparative genomics study in search of promising protein coding genes among the most conserved ones in reference fungal genomes. The CA3427 gene was selected on the basis of its presence among pathogenic fungi contrasting with its absence in the non pathogenic Saccharomyces cerevisiae. We report the crystal 3D-structure of the Candida albicans CA3427 protein at 2.1 Å resolution. The combined analysis of its sequence and structure reveals a structural fold originally associated with periplasmic binding proteins. The CA3427 structure highlights a binding site located between the two protein domains, corresponding to a sequence segment conserved among fungi. Two crystal forms of CA3427 were found, suggesting that the presence or absence of a ligand at the proposed binding site might trigger a “Venus flytrap” motion, coupled to the previously described activity of bacterial periplasmic binding proteins. The conserved binding site defines a new subfamily of periplasmic binding proteins also found in many bacteria of the bacteroidetes division, in a choanoflagellate (a free-living unicellular and colonial flagellate eukaryote) and in a placozoan (the closest multicellular relative of animals). A phylogenetic analysis suggests that this gene family originated in bacteria before its horizontal transfer to an ancestral eukaryote prior to the radiation of fungi. It was then lost by the Saccharomycetales which include Saccharomyces cerevisiae.