Epsilon Archive for Student Projects

Abstract

The purpose of this project was to investigate the structure-function relationship of two carbohydrate active enzymes, Endo-N-acetyl-β-(D)-glucosaminidase (EndoT) from Hypocrea jecorina and Cel5H from Saccharophagus degradans.

The enzyme Endo-T from H. jecorina is a highly specific endoglycosidase, which cleaves aspargine-linked mannose rich oligosaccharides, but not highly processed complex oligosaccharides from glycoproteins. Endo T cleaves the bond between two N-acetylglucosamine (GlcNAc) subunits found attached to glycosylated asparagine residue. The structure of native EndoT has in a previous study been determined. This crystal structure that then had been obtained was a structure with a zinc atom bound in the active site of the enzyme. To better understand the architecture of the active site of Endo-T, attempt was made in this study to determine the structure of a ligand-protein complex. For this purpose the protein was crystallized in the absence of zinc which otherwise could occupy the active site preventing the ligand from binding. Several ligands were tried in co-crystallization experiments, but no protein-ligand crystal complexes were obtained.

Cel5H from Saccharophagus degradans 2-40, a putative β-1,4 endoglucanase, belongs to glycoside hydrolase family 5. Very little is known about how this enzyme cleaves the substrate. In this study, we have tried to crystallize Cel5H with the aim to solve the three dimensional structure of the enzyme. Many attempts to crystallize the enzyme were performed, but no crystals were optained. To understand the substrate cleavage pattern of Cel5H, the function of enzyme was characterized by studying the hydrolysis pattern of the two substrates cellopentaose and cellohexose. The hydrolysis products were separated by reverse phase chromatography using a HPLC instrument. The results indicate that Cel5H has mainly endoglucanase activity and it has an preference for cleaving the substrate from the reducing end. The results were not conclusive enough here was not enough to if the enzyme was acting processively or not.