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Crystallization and X-ray diffraction analysis of ornithine cyclodeaminase from Pseudomonas putida
Shabnam Alam1, Susan C Wang, Frank J Ruzicka
1Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Summary
Ornithine cyclodeaminase (OCD) crystallization was achieved using high-throughput screening. This facilitates structural studies to understand enzyme catalysis and functional groups.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Ornithine cyclodeaminase (OCD) is a micro-crystallin family enzyme catalyzing L-ornithine to L-proline and ammonia conversion.
- Understanding OCD's catalytic mechanism requires elucidating its functional groups.
Purpose of the Study:
- To determine the crystal structure of Ornithine cyclodeaminase (OCD) from Pseudomonas putida.
- To identify enzyme functional groups critical for catalysis.
Main Methods:
- High-throughput crystallization screening using microbatch-under-oil technique.
- Optimization of crystallization conditions and structure determination via X-ray diffraction.
- Crystallization achieved using 40% 2-methyl-2,4-pentanediol, 0.1 M MES buffer at pH 6.0.
Main Results:
- Diffraction-quality crystals of OCD were obtained, diffracting X-rays to 1.68 A resolution.
- Crystals belong to space group P2(1)2(1)2(1) with specific unit-cell parameters.
- The asymmetric unit contains two molecules (38.5 kDa each), indicating 42% solvent content.
Conclusions:
- Successful crystallization of OCD provides a foundation for detailed structural analysis.
- Structural insights will elucidate the enzyme's catalytic mechanism and functional groups.
- Further structure determination using experimental phasing is in progress.