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

Acta Crystallographica. Section D, Biological Crystallography
C M HensgensBauke 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 (NH(4))(2)SO(4), 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 A and beta = 129.56 degrees. They diffract to 2.8 A resolution with X-rays from a rotating-anode generator.

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