Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS)

Deacetoxycephalosporin C synthase (DAOCS) is an iron(II) and 2-oxoglutarate -dependent oxygenase that catalyzes the conversion of penicillin N to deace toxycephalosporin C, the committed step in the biosynthesis of cephalospori n antibiotics. The crystal structure of DAOCS revealed that the C terminus of one molecule is inserted into the active site of its neighbor in a cycli cal fashion within a trimeric unit. This arrangement has hindered the gener ation of crystalline enzyme-substrate complexes. Therefore, we constructed a series of DAOCS mutants with modified C termini. Oxidation of 2-oxoglutar ate was significantly uncoupled from oxidation of the penicillin substrate in certain truncated mutants. The extent of uncoupling varied with the numb er of residues deleted and the penicillin substrate used. Crystal structure s were determined for the Delta R306 mutant complexed with iron(II) and 2-o xoglutarate (to 2.10 Angstrom) and the Delta R306A mutant complexed with ir on(II), succinate and unhydrated carbon dioxide (to 1.96 Angstrom). The lat ter may mimic a product complex, and supports proposals for a metal-bound CO2 intermediate during catalysis. (C) 2001 Academic Press.