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Strain relief at the active site of phosphoserine aminotransferase induced by radiation damage

¹ Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, 20521, Finland
² European Molecular Biology Laboratory (EMBL), Grenoble Outstation, Grenoble, Cedex 9, 38042, France
³ EMBL, Hamburg Outstation, c/o Deutsches Elektronen-Synchrotron (DESY), Hamburg, 22603, Germany

(RECEIVED February 7, 2005; FINAL REVISION March 10, 2005; ACCEPTED March 10, 2005)

The X-ray susceptibility of the lysine-pyridoxal-5'-phosphate Schiff base in Bacillus alcalophilus phosphoserine aminotransferase has been investigated using crystallographic data collected at 100 K to 1.3 Å resolution, complemented by on-line spectroscopic studies. X-rays induce deprotonation of the internal aldimine, changes in the Schiff base conformation, displacement of the cofactor molecule, and disruption of the Schiff base linkage between pyridoxal-5'-phosphate and the Lys residue. Analysis of the "undamaged" structure reveals a significant chemical strain on the internal aldimine bond that leads to a pronounced geometrical distortion of the cofactor. However, upon crystal exposure to the X-rays, the strain and distortion are relaxed and eventually diminished when the total absorbed dose has exceeded 4.7 x 10⁶ G. Our data provide new insights into the enzymatic activation of pyridoxal-5'-phosphate and suggest that special care should be taken while using macromolecular crystallography to study details in strained active sites.

Keywords: pyridoxal-5'-phosphate; Schiff base; phosphoserine aminotransferase; radiation damage

Abbreviations: AAT, aspartate aminotransferase • BALC, Bacillus alcalophilus • CD, circular dichroism • PLP, pyridoxal-5'-phosphate • PSAT, phosphoserine aminotransferase.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051397905.

Reprint requests to: Anastassios C. Papageorgiou, Turku Centre for Biotechnology, BioCity, Tykistökatu 6, Turku 20521, Finland; e-mail: apapageo{at}btk.fi; fax: +358-2-333-8000.

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