|
|
| Identifier |
uch.biology.phd//2004roidis |
| Title |
Κρυσταλλική δομή της ασεκυλοτρανσφερασης της χλωραμφενικόλης τύπου Ι από την E.Coli σε απονενζυμική μορφή και σε σύμπλοκο με τον αναστολέα φουσιδικό οξύ |
| Alternative Title |
Crystal structure of type I chloramphenicol acetyl transferase in apoenzyme form and complexed with inhibitor fusidic acid |
|
Author
|
Ροΐδης, Αλέξανδρος
|
| Abstract |
The phenomenon of antibiotic resistance in bacterial species is of major importance with clinical and ecological implications. Bacteria exhibit resistance to antibiotics via different pathways. Among these the enzymatic inactivation of antibiotics is of great interest. Resistance to chloramphenicol,a widely used antibiotic, is achieved via enzymatic acetylation of the primary hydroxyl group of the antibiotic compound.The acetyl group donor is acetyl coenzyme A. Acetylation of chloramphenicol occurs in many bacterial species due to the action of a family of evolutionary related enzymes which are chloramphenicol acetyltransferases. In Enterobacteria enzymatic acetylation of chloramphenicol is performed by three distinct variants form of the enzyme namely CATI, CATII and CATIII. Type I variant not only confers resistance to chloramphenicol but also to the widely used steroidal antibiotic fusidic acid, which acts as an inhibitor of the enzyme. Although CATI is inhibited by fusidic acid, by non reversible binding of the steroid to the active site, it confers resistance to the antibiotic by sequestering it from the cytoplasm. This extraordinary molecular selectivity of CATI is of great interest. In order to explore the mechanism of antibiotic resistance to chroramphenicol and to fusidic acid we have determined the crystal structure of type I chloramphenicol acetyltransferase CATI in apoenzyme form and complex with inhibitor fusidic acid. The three dimensional structure of CATI reveals a homotrimeric organization of the enzyme. The enzyme crystallizes in space group P21 and there are four trimers in the crystallographic asymmetric unit. The enzyme has a mixed α/β structure. The central structural element is an extended beta pleated sheet which spans the entire trimer molecular core and is surrounded by alpha helices which support the above mentioned sheet and make contacts with the solvent. The active sites of the enzyme are formed by amino acid residues belonging to adjacent subunits. Due to the homotrimeric structure of the enzyme there are three active sites, which can operate independently by random addition of the substrate and acetyl coenzyme A. The binding of fusidic acid is achieved through specific hydrogen bonding of the steroid molecule with the side chains of polar amino acid residues that protrude into the active site, as well as with ordered solvent molecules that occupy well defined space in the active site of the enzyme. The binding of the inhibitor is stabilized by non polar interactions with a number of hydrophobic amino acid residue mainly Phenylalanines which participate in π-stacking with the extended ring network of fusidic acid. The structure of the enzyme reveals complex non crystallographic symmetry between the four trimers. There are three twofolds local axis which relate the trimers by 1800 rotation. A calcium cation participates in the formation of one local axis. The chelating complex of the calcium cation consist of seven groups which adopt a pentagonal bipyramid geometry, which has been reported for other structures. Finally comparative analysis of the three dimensional structure of CATI reveals that the folding pattern common to chloramphenicol acetyltransferases is conserved in other functionally unrelated proteins which exhibit low levels of primary sequence homology with the afformentioned enzymes. Such findings suggests that mechanisms of folding conservation operate evolutionary and must be taken into account in the classification of protein structures. The structure of CATI complexed with fusidic acid not only explains the mechanism of resistance to this antibiotic but can also yield structural information for rational design of novel antibiotics based on fusidic acid that do not bind to CATI. Furthermore the mode of binding of fusidic yields novel insights in the architecture of steroid binding proteins.
|
| Language |
Greek |
| Subject |
Ένζυμα; Ακετυλοτρανσφεράση; Χλωραμφενικόλη; Φουσιδικό οξύ; Ανθεκτικότητα; Αντιβιοτικά; Κρυσταλλογραφία ακτίνων Χ |
| Issue date |
2004-03-11 |
| Date available |
2004-04-20 |
| Collection
|
School/Department--School of Sciences and Engineering--Department of Biology--Doctoral theses
|
|
|
Type of Work--Doctoral theses
|
| Views |
383 |
Κρυσταλλική δομή της ασεκυλοτρανσφερασης της χλωραμφενικόλης τύπου Ι από την E.Coli σε απονενζυμική μορφή και σε σύμπλοκο με τον αναστολέα φουσιδικό οξύ
