Obiettivo - Chitin is the second most abundantly distributed polysaccharide throughout nature. This homopolymer of N-acetyl-glucosamine is not only the major constituent of the fungal cell wall and the arthropod exoskeleton but also an important nutrient source of carbon and nitrogen in the marine environment. These enzymes are produced and secreted from chitinolytic bacteria and are Chitinases (EC.3.2.1.14) and Chitobiases (EC.3.2.1.30). Chitinases have been classified into families 18 and 19 of glycosyl hydrolases. They hydrolyse chitin to oligosaccharides of which N,N'-diacetyl-glucosamine is the predominant product. N,N'-diacetyl-glucosamine is the substrate for Chitobiase (trivial name for N-acetyl-glucosaminidase) which is classified into family 20 of glycosyl hydrolases.Specific objectives of the project are :- To solve the structure of the Serratia marcescens chitinase A;- To solve the structure of the Serratia marcescens chitobiase;- To determine the sequence of the gene coding for chitinase from Aeromonas caviae and perform biochemical characterisation of the protein;- To clone, overexpress, purify and crystallise the thermostable chitinase from Streptomyces thermoviolaceus.- 3D structure determination of Chitinase A from Serratia marcescens The structure of Chitinase A (ChiA) was solved by multiple isomorphous replacement and comprises three domains. The N-terminal domain (residues 24 to 137), which is made up of -beta-sheet, connects through a hinge region (residues 138 to 158) to the main alpha beta barrel domain (residues 159 to 442 and 517 to 563). The third domain, which has an alpha+beta fold, is formed by an insertion in the barrel motif (residues 443 to 516). The average B value for protein atoms is 24.1Å2. The N-terminal domain has a fold similar to that of the animal protein fibronectin type III (FnIII) module domains. Its function is yet unknown but might well facilitate the binding of the enzyme to the filamentous chitin substrate. The active site was identified by solving the structure of the enzyme with an oligomer of its natural substrate. The substrate binding site is formed by a long groove, located at the C terminal and of the beta strands of the alpha beta barrel. In all known enzymes with alpha/beta barrel structure, the active site is located at the end of the barrel. The active site residues are proposed to be Glu315 and possibly Asp391. Evidence for this is as follows :- site directed mutagenesis in the Bacillus circulans chitinase showed that the Glu204 to Gln mutation (Glu204 of Bacillus chitinase aligns with Glu315 of ChiA) decreased activity almost to zero;- Glu315 and Asp391 are completely conserved in bacterial chitinases;- the carboxylate oxygens of both residues are close to the C1 atom of the sugar ring. The quality of the complex does not allow us to make clear suggestions of the mode of substrate binding and for the structural features of the specificity of the chitin polysaccharide. Most probably the catalytic event occurs in a manner similar to that of lysozyme, i.e. general acid-base catalysis, with retention of configuration of the anomeric conformation of the C1 atom of the sugar ring. Currently, we are working on the structural elucidation of the complex of ChiA with its natural inhibitor allosamidin.- 3D structure determination of Chitobiase from Serratia marcescens The 3-D structure of Chitobiase was also solved by multiple isomorphous replacement. Chitobiase has an eight stranded alpha beta-barrel structure (domain III) surrounded by three additional domains :- domain I comprises residues 28 to 175. Two beta-pleated sheets wrap around a hydrophobic core. The motif starts with a three turn alpha-helix that points into solvent. Domain I is connected to domain II by a fifty amino acid long linker (residues 175 to 225) which folds around the alpha beta-barrel (domain III);- domain II (residues 225 to 334) shows two parallel helices and a seven stranded beta-sheet (partly parallel and partly antiparallel) faces the solvent. The beta-strands tilt about 30° to the helices. - domain III folds into an alpha beta-barrel motif. It comprises 465 amino acids (residues 340 to 815). Eight beta-strands inside and seven helices on the outside were found. The eighth helix is replaced by three helical segments and a beta-strand. The C-terminal end of the barrel faces towards domain I. The active site was identified by substrate and inhibitor binding studies to be at the C-terminus of the alpha beta barrel. Most prominent insertions ot the barrel motif are a loop towards domain I and two helices pointing into solvent. A long helix expands around the barrel and completes domain III. This helix has a kink after 4 turns where a glycine is found. Domain IV folds into two small beta-sheets. Based on the structure of the complex with the substrate disaccharide chitobiose and on previous biochemical data, an acid-base reaction mechanism is proposed in which only one protein carboxylate acts as catalyst, while the nucleophile is provided by the polar aceamido group of the sugar in a substrate assisted reaction, known as neighbouring group participation or anchimeric assistance. This is the first example of a natural substrate complex for a glycosyl hydrolase with a sugar in the +1 and -1 site on each side of the scissile bond. The reaction proceeds with retention of anomeric configuration. The catalytic domain of the homologous hexosaminidases is modelled on the structure of the catalytic alpha beta-barrel of chitobiase. Pathogenic mutations, previously classified by phenotype in the human Tay-Sachs and Sandhoff genetic diseases, are given a structural rationale.- Cloning, overexpression purification and characterisation of a thermophilic chitinase from Streptomyces thioviolaceus The chitinase gene chi40 was isolated from the thermophilic bacterium Streptomyces thioviolaceus cloned in pET-15b (fused with 6 His for affinity purification) and efficiently overexpressed in E. coli. The recombinant chitinase has a molecular weight of 40 kDa, it is highly active and shows significant thermostability. The melting temperature measured by CD spectroscopy was 74-75°C. Two forms of the enzyme were isolated showing alternative folds that were isolated and characterised. Both forms show very close biochemical properties but differ in their molecular fold. We are currently trying to crystallise both forms and to study the structural features of the protein in terms of thermostability and folding.- Cloning and primary structure of a chitinase from Aeromonas caviae A DNA fragment from the soil bacteria Aeromonas caviae containing the gene encoding an extracellular chitinase (Chi) has been cloned and sequenced. Computer analysis deduced an open reading frame encoding a protein of 865 amino acid (aa) sequence that shows high homology to the ChiA of Serratia marcescens. Expression in E. coli yielded enzymatically active protein with an estimated molecular weight of 94 kd. The deduced aa sequence is 23 aa longer at the amino terminus than that determined experimentally by sequencing of the purified protein, suggesting that a leader sequence is removed during transport of the enzyme across the cell membrane. The C-terminus extension found in the chitinase from Aeromonas caviae is larger than the chitinase from Alteromonas sp. The C-terminus contains two small related sequences that probably arose by gene duplication. This domain also aligns with the last 40 residues of two more Bacillus cellulase gene products (CELA and CELB). These observations suggest to us that the C-terminal region of the Aeromonas caviae chitinase and the Bacillus sp. strain N-4 cellulases are functionally related and may be involved in the ability of these enzymes to degrade their highly hydrophobic substrates. Major scientific breakthroughs are :- the first crystal structure of the Chitinase A from Serratia marcescens;- the first crystal structure of the Chitobiase from Serratia marcescens;- the use of structural information from prokaryotic organisms to model the structure of a eucaryotic enzyme using a new algorithm. This novel approach helped us to give a working model on the structural basis of Tay-Sachs and Sandhoff. Programma(i) IC-ISC C - Activity (Euratom, EEC) on cooperation in science and technology, 1984- Argomento(i) Data not available Invito a presentare proposte Data not available Meccanismo di finanziamento CSC - Cost-sharing contracts Coordinatore EUROPEAN MOLECULAR BIOLOGY LABORATORY Contributo UE Nessun dato Indirizzo Meyerhofstrasse 1 HEIDELBERG Germania Mostra sulla mappa Costo totale Nessun dato
