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

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.

Substructure solution withSHELXD

“…The effect of different resolution cutoffs on the success rate of SHELXD in solving the AT substructure (Hensgens et al, 2002). The correlation coef®cient between the signed |F hkl | À |F hkl | differences of the high-energy remote and peak-wavelength data (CC ano ) is shown in green.…”

“…3. In the case of AT (Hensgens et al, 2002), SHELXD did not ®nd any solutions when any data to higher resolution than 3.5 A Ê were included. For different cutoffs between 3.5 and 4.5 A Ê , the quality of the best solution as measured by the highest correlation coef®cient achieved (CC max ) in a run of 100 tries is very similar.…”

Structures of an Isopenicillin N Converting Ntn-Hydrolase Reveal Different Catalytic Roles for the Active Site Residues of Precursor and Mature Enzyme

“…Like other Ntn-hydrolases, AT is synthesized as a precursor enzyme that is proteolytically cleaved to yield the active enzyme. Mutants of Cys103 are not cleaved and the Cys103Ala mutant protein is therefore considered a good model of precursor AT (Hensgens et al, 2002;Tobin et al, 1995). Its structure shows that the molecule consists of a single chain, with the peptide bond between residues 102 and 103 intact (Figure 2A).…”

Three-dimensional structures of enzymes useful for ?-lactam antibiotic production

“…As this enzyme accepts a wide variety of sidechains, it might become a very useful tool in antibiotic production and structural studies are under way [9].…”