IngentaConnect Crystallization and X-ray diffraction analysis of ornithine cyclo...

Authors: Alam, Shabnam; Wang, Susan C.; Ruzicka, Frank J.; Frey, Perry A.; Wedekind, Joseph E.

Source: Acta Crystallographica Section D, Volume 60, Number 5, May 2004 , pp. 941-944(4)

Abstract:

Ornithine cyclodeaminase (OCD) is a member of the μ-crystallin protein family, the biological activity of which is the conversion ofl-ornithine tol-proline and ammonia. In order to elucidate the functional groups of this enzyme that are involved in catalysis, the crystallization of OCD from Pseudomonas putida was undertaken. Using microbatch-under-oil screening at the high-throughput crystallization laboratory (HTC) at the Hauptman-Woodward Medical Research Institute Inc. (HWI Buffalo, NY, USA), numerous crystallization conditions were rapidly identified. Several conditions could be reproduced on a larger scale as vapor-diffusion experiments in-house. The best diffraction-quality crystals were obtained from solutions of 40%(v/v) 2-methyl-2,4-pentanediol buffered at pH 6.0 with 0.1 M MES and diffracted X-rays to 1.68 Å resolution. Crystals belonged to space group P2₁2₁2₁, with unit-cell parameters a = 70.0, b = 78.3, c = 119.4 Å. The V_M was 2.1 Å³ Da⁻¹, corresponding to 42% solvent, which is consistent with two 38.5 kDa molecules per asymmetric unit. The structure determination is under way using experimental phasing methods.

Keywords: nitrogen metabolism; μ-crystallin; high-throughput crystallization; dehydrogenase

Document Type: Research article

DOI: 10.1107/S0907444904005256

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Crystallization and X-ray diffraction analysis of ornithine cyclodeaminase from Pseudomonas putida