The last step in cephalosporin C formation revealed: crystal structures of deacetylcephalosporin C acetyltransferase from Acremonium chrysogenum in complexes with reaction intermediates.
Deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of cephalosporin C, a broad-spectrum beta-lactam antibiotic of large clinical importance. The acetyl transfer step has been suggested to be limiting for cephalosporin C biosynthesis, but has so far escaped detailed structural analysis. We present here the crystal structures of DAC-AT in complexes with reaction intermediates, providing crystallographic snapshots of the reaction mechanism. The enzyme is found to belong to the alpha/beta hydrolase class of acetyltransferases, and the structures support previous observations of a double displacement mechanism for the acetyl transfer reaction in other members of this class of enzymes. The structures of DAC-AT reported here provide evidence of a stable acyl-enzyme complex, thus underpinning a mechanism involving acetylation of a catalytic serine residue by acetyl coenzyme A, followed by transfer of the acetyl group to deacetylcephalosporin C through a suggested tetrahedral transition state.
Lejon
Sara
S
Department of Cell and Molecular Biology, Uppsala University, Biomedical Centre, Box 596, S-751 24 Uppsala, Sweden.
Ellis
Jacqueline
J
Valegård
Karin
K
D000107:Acetylation; D000123:Acetyltransferases / Q000737:chemistry*; D000164:Acremonium / Q000201:enzymology*; D001665:Binding Sites; D002511:Cephalosporins / Q000096:biosynthesis / Q000737:chemistry*; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry
D002511:Cephalosporins; D005656:Fungal Proteins; D021122:Protein Subunits; C025163:cephalosporin C; D000123:Acetyltransferases; C018707:acetyl CoA-deacetylcephalosporin C acetyltransferase