Genome and Transcriptome of Clostridium phytofermentans, Catalyst for the Direct Conversion of Plant Feedstocks to Fuels.

Clostridium phytofermentans was isolated from forest soil and is distinguished by its capacity to directly ferment plant cell wall polysaccharides into ethanol as the primary product, suggesting that it possesses unusual catabolic pathways. The objective of the present study was to understand the molecular mechanisms of biomass conversion to ethanol in a single organism, Clostridium phytofermentans, by analyzing its complete genome and transcriptome during growth on plant carbohydrates. The saccharolytic versatility of C.

Crystal structure of Bacillus subtilis SPP1 phage gp22 shares fold similarity with a domain of lactococcal phage p2 RBP.

SPP1 is a siphophage infecting the gram-positive bacterium Bacillus subtilis. It is constituted by an icosahedric head and a long non-contractile tail formed by gene products (gp) 17-21. A group of 5 small genes (gp 22-24.

The XcpV/GspI pseudopilin has a central role in the assembly of a quaternary complex within the T2SS pseudopilus.

Gram-negative bacteria use the sophisticated type II secretion system (T2SS) to secrete a large number of exoproteins into the extracellular environment. Five proteins of the T2SS, the pseudopilins GspG-H-I-J-K, are proposed to assemble into a pseudopilus involved in the extrusion of the substrate through the outer membrane channel. Recent structural data have suggested that the three pseudopilins GspI-J-K are organized in a trimeric complex located at the tip of the GspG-containing pseudopilus.

Structure and function of phage p2 ORF34(p2), a new type of single-stranded DNA binding protein.

Lactococcus lactis, a Gram-positive bacterium widely used by the dairy industry, is subject to infection by a diverse population of virulent phages, predominantly by those of the 936 group, including the siphovirus phage p2. Confronted with the negative impact of phage infection on milk fermentation, the study of the biology of lactococcal provides insight from applied and fundamental perspectives. We decided to characterize the product of the orf34 gene from lactococcus phage p2, which was considered as a candidate single-stranded DNA binding protein (SSB) due to its localization downstream of a gene coding for a single-strand annealing protein.

Production and biophysical characterization of the CorA transporter from Methanosarcina mazei.

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 recombination, (ii) high-throughput expression screening, (iii) fermenter-based large-scale production, (iv) high-throughput detergent solubilization screening, (v) protein purification, (vi) multiangle static light scattering/refractometry characterization of purified proteins, (vii) circular dichroism spectroscopy, and (viii) detergent concentration measurements by Fourier transform infrared (FT-IR) spectroscopy.

Green fluorescent protein and factorial approach: an effective partnership for screening the soluble expression of recombinant proteins in Escherichia coli.

We report how the combined use of protein expression reporter green fluorescent protein (GFP), and of an incomplete factorial approach ("InFFact") made of 12 combinations of different states of three expression variables (bacterial strains, culture media and expression temperatures) created a convenient tool for screening the soluble expression of recombinant proteins in Escherichia coli (E. coli). In the first part of this work, we used two recombinant proteins that could be easily detected by Western blotting in the soluble fraction of E.

Solution structure of PcFK1, a spider peptide active against Plasmodium falciparum.

Psalmopeotoxin I (PcFK1) is a 33-amino-acid residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. It has been recently shown to possess strong antiplasmodial activity against the intra-erythrocyte stage of Plasmodium falciparum in vitro. Although the molecular target for PcFK1 is not yet determined, this peptide does not lyse erythrocytes, is not cytotoxic to nucleated mammalian cells, and does not inhibit neuromuscular function.

Structural insights into the mechanism of formation of cellulosomes probed by small angle X-ray scattering.

Exploring the mechanism by which the multiprotein complexes of cellulolytic organisms, the cellulosomes, attain their exceptional synergy is a challenge for biologists. We have studied the solution structures of the Clostridium cellulolyticum cellulosomal enzyme Cel48F in the free and complexed states with cohesins from Clostridium thermocellum and Clostridium cellulolyticum by small angle x-ray scattering in order to investigate the conformational events likely to occur upon complexation. The solution structure of the free cellulase indicates that the dockerin module is folded, whereas the linker connecting the catalytic module to the dockerin is extended and flexible.

Crystal structure of E.coli alcohol dehydrogenase YqhD: evidence of a covalently modified NADP coenzyme.

In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame (ORF) products of unknown function, we have determined the structure of YqhD at 2.0A resolution using the single wavelength anomalous diffraction method at the Pt edge. The crystal structure of YqhD reveals that it is an NADP-dependent dehydrogenase, a result confirmed by activity measurements with several alcohols.

Crystal structure and reactivity of YbdL from Escherichia coli identify a methionine aminotransferase function.

The ybdL gene of Escherichia coli codes for a protein of unknown function. Sequence analysis showed moderate homology to several vitamin B(6) dependent enzymes, suggesting that it may bind pyridoxal-5'-phosphate. The structure analysis of YbdL to 2.

Domain swapping of a llama VHH domain builds a crystal-wide beta-sheet structure.

Among mammals, camelids have a unique immunological system since they produce functional antibodies devoid of light chains and CH1 domains. To bind antigens, whether they are proteins or haptens, camelids use the single domain VH from their heavy chain (VHH). We report here on such a llama VHH domain (VHH-R9) which was raised against a hapten, the RR6 red dye.

The Escherichia coli YadB gene product reveals a novel aminoacyl-tRNA synthetase like activity.

In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame products of unknown function, we have determined the structure of YadB at 1.5A using molecular replacement. The YadB protein is 298 amino acid residues long and displays 34% sequence identity with E.

Solution structure of ADO1, a toxin extracted from the saliva of the assassin bug, Agriosphodrus dohrni.

ADO1 is a toxin purified from the saliva of the assassin bug, Agriosphodrus dohrni. Because of its similarity in sequence to Ptu1 from another assassin bug, we did not assess its pharmacologic target. Here, we demonstrate by electrophysiologic means that ADO1 targets the P/Q-type voltage-sensitive calcium channel.

The Y181C substitution in 3'-azido-3'-deoxythymidine-resistant human immunodeficiency virus, type 1, reverse transcriptase suppresses the ATP-mediated repair of the 3'-azido-3'-deoxythymidine 5'-monophosphate-terminated primer.

Resistance to zidovudine (3'-azido-3'-deoxythymidine, AZT) by the human immunodeficiency virus, type 1, requires multiple amino acid substitutions such as D67N/K70R/T215F/K219Q in the viral reverse transcriptase (RT). In this background of AZT resistance, additional "suppressive" substitutions such as Y181C restore sensitivity to AZT. In order to characterize the mechanism of this AZT resistance suppression, the Y181C substitution was introduced into both wild-type and AZT-resistant reverse transcriptase.

An evolving hierarchical family classification for glycosyltransferases.

Glycosyltransferases are a ubiquitous group of enzymes that catalyse the transfer of a sugar moiety from an activated sugar donor onto saccharide or non-saccharide acceptors. Although many glycosyltransferases catalyse chemically similar reactions, presumably through transition states with substantial oxocarbenium ion character, they display remarkable diversity in their donor, acceptor and product specificity and thereby generate a potentially infinite number of glycoconjugates, oligo- and polysaccharides. We have performed a comprehensive survey of glycosyltransferase-related sequences (over 7200 to date) and present here a classification of these enzymes akin to that proposed previously for glycoside hydrolases, into a hierarchical system of families, clans, and folds.

Dimension, shape, and conformational flexibility of a two domain fungal cellulase in solution probed by small angle X-ray scattering.

Cellulase Cel45 from Humicola insolens has a modular structure with a catalytic module and a cellulose-binding module (CBM) separated by a 36 amino acid, glycosylated, linker peptide. The solution conformation of the entire two domain Cel45 protein as well as the effect of the length and flexibility of the linker on the spatial arrangement of the constitutive modules were studied by small angle x-ray scattering combined with the known three-dimensional structure of the individual modules. The measured dimensions of the enzyme show that the linker exhibits an extended conformation leading to a maximum extension between the two centers of mass of each module corresponding to about four cellobiose units on a cellulose chain.

X-ray structure and ligand binding study of a moth chemosensory protein.

Chemosensory proteins (CSPs) are believed to be involved in chemical communication and perception. Such proteins, of M(r) 13,000, have been isolated from several sensory organs of a wide range of insect species. Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae.

Solution structure and backbone dynamics of an antigen-free heavy chain variable domain (VHH) from Llama.

Camelids, (dromedaries, camels, and llamas) produce heavy-chains antibodies, with their antigen recognition sites composed of a single VH-like domain, referred to as VHH. The solution structure of one of these VHHs domains (VHH-H14), raised against the alpha subunit of the human chorionic gonadotropin hormone (hCG), has been determined by (15)N heteronuclear three-dimensional NMR spectroscopy. The framework is well resolved within the set of 20 best-calculated NMR structures and is close to that of classical VH domains from vertebrate antibodies, consisting of two antiparallel beta-sheets organized in a beta-barrel.

A peptide mimic of an antigenic loop of alpha-human chorionic gonadotropin hormone: solution structure and interaction with a llama V(HH) domain.

The X-ray structure of a ternary complex between human chorionic gonadotropin hormone (hCG) and two Fvs recognizing its alpha and beta subunits has been recently determined. The Fvs recognize the elongated hCG molecule by its two ends, one being the Leu-12-Cys-29 loop of the alpha subunit. We have designed and synthesized a 17-amino-acid peptide (named PepH14) derived from the sequence of this antigenic loop with the purpose of mimicking its three-dimensional structure and its affinity for antibodies.

Solution structure of Ptu1, a toxin from the assassin bug Peirates turpis that blocks the voltage-sensitive calcium channel N-type.

Ptu1 is a toxin from the assassin bug Peirates turpis which has been demonstrated to bind reversibly the N-type calcium channels and to have lower affinity than the omega-conotoxin MVIIA. We have determined the solution structure of Ptu1 by use of conventional two-dimensional NMR techniques followed by distance-geometry and molecular dynamics. The calculated structure of Ptu1 belongs to the inhibitory cystin knot structural family (ICK) that consists of a compact disulfide-bonded core from which four loops emerge.

Domain swing upon His to Ala mutation in nitrite reductase of Pseudomonas aeruginosa.

The nitrite reductase (NIR) from Pseudomonas aeruginosa (NIR-Pa) is a soluble enzyme catalysing the reduction of nitrite (NO2(-)) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which the monomers carry a c-heme domain and a d(1)-heme domain. The structures of the enzyme in both the oxidised and reduced state were solved previously and indicate His327 and His369 as putative catalytic residues.

A census of carbohydrate-active enzymes in the genome of Arabidopsis thaliana.

The synthesis, modification, and breakdown of carbohydrates is one of the most fundamentally important reactions in nature. The structural and functional diversity of glycosides is mirrored by a vast array of enzymes involved in their synthesis (glycosyltransferases), modification (carbohydrate esterases) and breakdown (glycoside hydrolases and polysaccharide lyases). The importance of these processes is reflected in the dedication of 1-2% of an organism's genes to glycoside hydrolases and glycosyltransferases alone.

Chemosensory protein from the moth Mamestra brassicae. Expression and secondary structure from 1H and 15N NMR.

A group of ubiquitous small proteins (average 13 kDa) has been isolated from several sensory organs of a wide range of insect species. They are believed to be involved in chemical communication and perception (olfaction or taste) and have therefore been called chemo-sensory proteins (CSPs). Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae.

Lateral recognition of a dye hapten by a llama VHH domain.

Camelids, camels and llamas, have a unique immune system able to produce heavy-chain only antibodies. Their VH domains (VHHs) are the smallest binding units produced by immune systems, and therefore suitable for biotechnological applications through heterologous expression. The recognition of protein antigens by these VHHs is rather well documented, while less is known about the VHH/hapten interactions.