Acta Crystallographica Section D

Biological Crystallography

Volume 58, Part 4 (April 2002)

crystallization papers

Acta Cryst. (2002). D58, 716-718 [ doi:10.1107/S0907444902003244 ]

Purification, crystallization and preliminary X-ray diffraction of Cys103Ala acyl coenzyme A:isopenicillin N acyltransferase from Penicillium chrysogenum

C. M. H. Hensgens, E. A. Kroezinga, B. A. van Montfort, J.-M. van der Laan, J. D. Sutherland and B. W. Dijkstra

Abstract: Penicillins and cephalosporins are an efficacious group of antibiotics produced by fungi such as Penicillium chrysogenum and Acremonium chrysogenum. The last step in their biosynthesis is catalyzed by acyl coenzyme A:isopenicillin N transferase (AT). This enzyme is produced as a single-chain proenzyme, which is activated by autocatalytic cleavage of the Gly102-Cys103 peptide bond, resulting in a heterodimeric protein with subunits of 11 and 29 kDa. The Cys103Ala mutant of the proenzyme, which does not undergo this cleavage, was purified and crystallized. Diffraction-quality crystals of the mutant and an L-SeMet-substituted mutant were obtained by vapour diffusion against solutions containing (NH₄)₂SO₄, NaCl and HEPES-NaOH pH 7.5. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 231.36, b = 68.27, c = 151.31 Å and [beta] = 129.56°. They diffract to 2.8 Å resolution with X-rays from a rotating-anode generator.

Keywords: acyl coenzyme A:isopenicillin N acyltransferase; penicillin biosynthesis.