Structural studies of Erwinia carotovora L-Asparaginase by X-ray crystallography

Abstract: Bacterial L-asparaginases (E.C.3.5.1.1) are enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid. For the past 30 years these enzymes have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. The presence of a low rate glutaminase activity however causes serious side-effects to patients in treatment, as glutamine depletion give rise to neurotoxicity, anaphylaxis, and other hypersensitivity reactions. The interest in the enzyme from Erwinia carotovora originates from the fact that it shows a decreased glutaminase activity, and therefore the enzyme is expected to exhibit fewer side effects when used in therapy. The main focus of this thesis is the crystal structure determination of L-asparaginase from Erwinia carotovora in the presence of aspartic acid at 2.5 Å resolution. The structure was refined to an R/Rfree factor of 19.9/28.6 with good stereochemistry. L-Asparaginases are homotetrameric enzymes with a known 222 symmetry and an identical fold. The Erwinia carotovora asparaginase consists of eight monomers of 330 amino acid residues each. In this case the enzyme is active as a dimer of tetramers. The two tetramers have an inner twofold non-crystallographic symmetry. Each monomer forms two identifiable domains a large N-domain and a small C-domain. The active sites are found at a topological switch-point between those domains.