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

Bacterial L-asparaginases (E.C. 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….


1 Introduction
1.1 Background
1.1.1 Therapeutic enzymes
1.1.2 L-Asparaginase
1.2 Structure determination methods
1.2.1 X-ray crystallography
1.2.2 Nuclear magnetic resonance
2 Experimental approach
2.1 Crystal characterization
2.1.1 Theorem: Bragg’s Law
2.1.2 Matthews coecient
2.2 Diraction pattern
2.2.1 Indexing and scaling
2.3 Phase problem and determination
2.3.1 Isomorphous replacement
2.3.2 Molecular Replacement
2.3.3 Multiple-wavelength Anomalous Diraction
2.4 Structure visualization
2.4.1 The R-factor
2.4.2 Dierence map
2.4.3 Electron density map
2.4.4 Omit map
3 Experimental details
3.1 Crystallization
3.2 Molecular Replacement for ErCAR
3.3 Renement
3.3.1 The B-factor
3.4 Rebuilding
3.4.1 Water molecules
4 Results
4.1 Quality of the structure
4.2 Data collection and reffnement statistics
4.3 Structural features of L-asparaginase
4.3.1 The monomer structure
4.3.2 Description of the active site
4.3.3 The tetramer structure
4.3.4 The octamer structure
5 Discussion
6 Conclusion

Author: Andersson, Charlotta

Source: Linköping University

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