Biomedical subjects

Alain Roussel

Publications and source records attributed to Alain Roussel.

6 recordsLinked to original sources

PubMed2003

Structural analysis of xylanase inhibitor protein I (XIP-I), a proteinaceous xylanase inhibitor from wheat (Triticum aestivum, var. Soisson).

A novel class of proteinaceous inhibitors exhibiting specificity towards microbial xylanases has recently been discovered in cereals. The three-dimensional structure of xylanase inhibitor protein I (XIP-I) from wheat (Triticum aestivum, var. Soisson) was determined by X-ray crystallography at 1.8 A (1 A=0.1 nm) resolution. The inhibitor possesses a (beta/alpha)(8) barrel fold and has structural features typical of glycoside hydrolase family 18, namely two consensus regions, approximately corresponding to the third and fourth barrel strands, and two non-proline cis -peptide bonds, Ser(36)-Phe and Trp(256)-Asp (in XIP-I numbering). However, detailed structural analysis of XIP-I revealed several differences in the region homologous with the active site of chitinases. The catalytic glutamic acid residue of family 18 chitinases [Glu(127) in hevamine, a chitinase/lysozyme from the rubber tree (Hevea brasiliensis)] is conserved in the structure of the inhibitor (Glu(128)), but its side chain is fully engaged in salt bridges with two neighbouring arginine residues. Gly(81), located in subsite -1 of hevamine, where the reaction intermediate is formed, is replaced by Tyr(80) in XIP-I. The tyrosine side chain fills the subsite area and makes a strong hydrogen bond with the side chain of Glu(190) located at the opposite side of the cleft, preventing access of the substrate to the catalytic glutamic acid. The structural differences in the inhibitor cleft structure probably account for the lack of activity of XIP-I towards chitin.

Amino Acid Sequence↗

PubMed2002

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. In ten months, 25 proteins were subjected to crystallization; 13 yielded crystals, of which ten led to usable data sets and five to structures. Furthermore, the results using a robot dispensing 50-200 nl drops indicate that smaller protein samples can be used for crystallization. These still partial results might indicate present and future directions for those who have to make crucial choices concerning their crystallization platform in structural genomics programs.

Crystallization↗

PubMed2002

Specific characterization of substrate and inhibitor binding sites of a glycosyl hydrolase family 11 xylanase from Aspergillus niger.

The importance of aromatic and charged residues at the surface of the active site of a family 11 xylanase from Aspergillus niger was evaluated using site-directed mutagenesis. Ten mutant proteins were heterologously produced in Pichia pastoris, and their biochemical properties and kinetic parameters were determined. The specific activity of the Y6A, Y10A, Y89A, Y164A, and W172A mutant enzymes was drastically reduced. The low specific activities of Y6A and Y89A were entirely accounted for by a change in k(cat) and K(m), respectively, whereas the lower values of Y10A, Y164A, and W172A were due to a combination of increased K(m) and decreased k(cat). Tyr(6), Tyr(10), Tyr(89), Tyr(164), and Trp(172) are proposed as substrate-binding residues, a finding consistent with structural sequence alignments of family 11 xylanases and with the three-dimensional structure of the A. niger xylanase in complex with the modeled xylobiose. All other variants, D113A, D113N, N117A, E118A, and E118Q, retained full wild-type activity. Only N117A lost its sensitivity to xylanase inhibitor protein I (XIP-I), a protein inhibitor isolated from wheat, and this mutation did not affect the fold of the xylanase as revealed by circular dichroism. The N117A variant showed kinetics, pH stability, hydrolysis products pattern, substrate specificity, and structural properties identical to that of the wild-type xylanase. The loss of inhibition, as measured in activity assays, was due to abolition of the interaction between XIP-I and the mutant enzyme, as demonstrated by surface plasmon resonance and electrophoretic titration. A close inspection of the three-dimensional structure of A. niger xylanase suggests that the binding site of XIP-I is located at the conserved "thumb" hairpin loop of family 11 xylanases.

Amino Acid Sequence↗

PubMed2002

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. One of them, CSPMbraA6, a 112-amino acid antennal protein, has been expressed in large quantities and is soluble in the Escherichia coli periplasm. X-ray structure determination has been performed in parallel with ligand binding assays using tryptophan fluorescence quenching. The protein has overall dimensions of 25 x 30 x 32 A and exhibits a novel type of alpha-helical fold with six helices connected by alpha-alpha loops. A narrow channel extends within the protein hydrophobic core. Fluorescence quenching with brominated alkyl alcohols or fatty acids and modeling studies indicates that CSPMbraA6 is able to bind such compounds with C12-18 alkyl chains. These ubiquitous proteins might have the role of extracting hydrophobic linear compounds (pheromones, odors, or fatty acids) dispersed in the phospholipid membrane and transporting them to their receptor.

Amino Acid Sequence↗

PubMed2002

A hyperthermostable D-ribose-5-phosphate isomerase from Pyrococcus horikoshii characterization and three-dimensional structure.

A gene homologous to D-ribose-5-phosphate isomerase (EC 5.3.1.6) was found in the genome of Pyrococcus horikoshii. D-ribose-5-phosphate isomerase (PRI) is of particular metabolic importance since it catalyzes the interconversion between the ribose and ribulose forms involved in the pentose phosphate cycle and in the process of photosynthesis. The gene consisting of 687 bp was overexpressed in Escherichia coli, and the resulting enzyme showed activity at high temperatures with an optimum over 90 degrees C. The crystal structures of the enzyme, free and in complex with D-4-phosphoerythronic acid inhibitor, were determined. PRI is a tetramer in the crystal and in solution, and each monomer has a new fold consisting of two alpha/beta domains. The 3D structures and the characterization of different mutants indicate a direct or indirect catalytic role for the residues E107, D85, and K98.

Aldose-Ketose Isomerases↗

PubMed2001

Crystal structure of the open form of dog gastric lipase in complex with a phosphonate inhibitor.

Fat digestion in humans and some mammals such as dogs requires the successive intervention of two lipases: gastric lipase, which is stable and active despite the highly acidic stomach environment, followed by the classical pancreatic lipase secreted into the duodenum. We previously solved the structure of recombinant human gastric lipase (HGL) at 3.0-A resolution in its closed form; this was the first structure to be described within the mammalian acid lipase family. Here we report on the open structure of the recombinant dog gastric lipase (r-DGL) at 2.7-A resolution in complex with the undecyl-butyl (C11Y4) phosphonate inhibitor. HGL and r-DGL show 85.7% amino acid sequence identity, which makes it relevant to compare the forms from two different species. The open r-DGL structure confirms the previous description of the HGL catalytic triad (Ser(153), His(353), and Asp(324)) with the catalytic serine buried and an oxyanion hole (NH groups of Gln(154) and Leu(67)). In r-DGL, the binding of the C11Y4 phosphonate inhibitor induces part of the cap domain, the lid, to roll over the enzyme surface and to expose a catalytic crevice measuring approximately 20 x 20 x 7 A(3). The C11Y4 phosphonate fits into this crevice, and a molecule of beta-octyl glucoside fills up the crevice. The C11Y4 phosphonate inhibitor and the detergent molecule suggest a possible binding mode for the natural substrates, the triglyceride molecules.

Animals↗