Structural insights into marine carbohydrate degradation by family GH16 κ-carrageenases.

Carrageenans are sulfated α-1,3-β-1,4-galactans found in the cell wall of some red algae that are practically valuable for their gelation and biomimetic properties but also serve as a potential carbon source for marine bacteria. Carbohydrate degradation has been studied extensively for terrestrial plant/bacterial systems, but sulfation is not present in these cases, meaning the marine enzymes used to degrade carrageenans must possess unique features to recognize these modifications. To gain insights into these features, we have focused on κ-carrageenases from two distant bacterial phyla, which belong to glycoside hydrolase family 16 and cleave the β-1,4 linkage of κ-carrageenan.

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.

Crystal structure and function of a DARPin neutralizing inhibitor of lactococcal phage TP901-1: comparison of DARPin and camelid VHH binding mode.

Combinatorial libraries of designed ankyrin repeat proteins (DARPins) have been proven to be a valuable source of specific binding proteins, as they can be expressed at very high levels and are very stable. We report here the selection of DARPins directed against a macromolecular multiprotein complex, the baseplate BppUxBppL complex of the lactococcal phage TP901-1. Using ribosome display, we selected several DARPins that bound specifically to the tip of the receptor-binding protein (RBP, the BppL trimer).

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.

Post-genomic insights into the plant polysaccharide degradation potential of Aspergillus nidulans and comparison to Aspergillus niger and Aspergillus oryzae.

The plant polysaccharide degradative potential of Aspergillus nidulans was analysed in detail and compared to that of Aspergillus niger and Aspergillus oryzae using a combination of bioinformatics, physiology and transcriptomics. Manual verification indicated that 28.4% of the A.

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.

VaZyMolO: a tool to define and classify modularity in viral proteins.

Viral structural genomic projects aim at unveiling the function of unknown viral proteins by employing high-throughput approaches to determine their 3D structure and to identify their function through fold-homology studies. The 'viral enzyme module localization' (VaZyMolO) tool has been developed, which aims at defining viral protein modules that might be expressed in a soluble and functionally active form, thereby identifying candidates for crystallization studies. VaZyMolO includes 114 complete viral genome sequences of both negative- and positive-sense, single-stranded RNA viruses available from NCBI.

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.

Structure of Escherichia coli YhdH, a putative quinone oxidoreductase.

As part of a structural genomics project on bacterial gene products of unknown function, the crystal structures of YhdH, a putative quinone oxidoreductase, and its complex with NADP have been determined at 2.25 and 2.6 A resolution, respectively.

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 crystal structure of the Escherichia coli lipocalin Blc suggests a possible role in phospholipid binding.

Lipocalins form a large multifunctional family of small proteins (15-25 kDa) first discovered in eukaryotes. More recently, several types of bacterial lipocalins have been reported, among which Blc from Escherichia coli is an outer membrane lipoprotein. As part of our structural genomics effort on proteins from E.

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.

Crystal structure of Thermotoga maritima alpha-L-fucosidase. Insights into the catalytic mechanism and the molecular basis for fucosidosis.

Fucosylated glycoconjugates are involved in numerous biological events, and alpha-l-fucosidases, the enzymes responsible for their processing, are therefore of crucial importance. Deficiency in alpha-l-fucosidase activity is associated with fucosidosis, a lysosomal storage disorder characterized by rapid neurodegeneration, resulting in severe mental and motor deterioration. To gain insight into alpha-l-fucosidase function at the molecular level, we have determined the crystal structure of Thermotoga maritima alpha-l-fucosidase.

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.

Structural genomics of the SARS coronavirus: cloning, expression, crystallization and preliminary crystallographic study of the Nsp9 protein.

The aetiologic agent of the recent epidemics of Severe Acute Respiratory Syndrome (SARS) is a positive-stranded RNA virus (SARS-CoV) belonging to the Coronaviridae family and its genome differs substantially from those of other known coronaviruses. SARS-CoV is transmissible mainly by the respiratory route and to date there is no vaccine and no prophylactic or therapeutic treatments against this agent. A SARS-CoV whole-genome approach has been developed aimed at determining the crystal structure of all of its proteins or domains.

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.

The crystal structure of the Escherichia coli YfdW gene product reveals a new fold of two interlaced rings identifying a wide family of CoA transferases.

Because of its toxicity, oxalate accumulation from amino acid catabolism leads to acute disorders in mammals. Gut microflora are therefore pivotal in maintaining a safe intestinal oxalate balance through oxalate degradation. Oxalate catabolism was first identified in Oxalobacter formigenes, a specialized, strictly anaerobic bacterium.

A medium-throughput crystallization approach.

The first results of a medium-scale structural genomics program clearly demonstrate the value of using a medium-throughput crystallization approach based on a two-step procedure: a large screening step employing robotics, followed by manual or automated optimization of the crystallization conditions. The structural genomics program was based on cloning in the Gateway vectors pDEST17, introducing a long 21-residue tail at the N-terminus. So far, this tail has not appeared to hamper crystallization.

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.