Studies on the Active Site of Deacetoxycephalosporin C Synthase

The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25% of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel filtration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS was crystallized ±Fe(II) and/or 2-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 Å resolution and belonged to the R3 space group (unit cell dimensions: a = b = 106.4 Å, c = 71.2 Å; α = β = 90°, γ = 120° (in the hexagonal setting)). Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives. X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry defined by the crystal structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II). For Fe(II) in DAOCS the best fit to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 Å, three nitrogen or oxygen atoms at 2.11 Å and one other light atom at 2.04 Å. For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 Å), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 Å, another O atom at 2.08 Å and one at 2.03 Å. Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism.