The structural basis of cephalosporin formation in a mononuclear ferrous enzyme

Abstract

Deacetoxycephalosporin-C synthase (DAOCS) is a mononuclear ferrous enzyme that transforms penicillins into cephalosporins by inserting a carbon atom into the penicillin nucleus. In the first half-reaction, dioxygen and 2-oxoglutarate produce a reactive iron-oxygen species, succinate and CO2. The oxidizing iron species subsequently reacts with penicillin to give cephalosporin and water. Here we describe high-resolution structures for ferrous DAOCS in complex with penicillins, the cephalosporin product, the cosubstrate and the coproduct. Steady-state kinetic data, quantum-chemical calculations and the new structures indicate a reaction sequence in which a 'booby-trapped' oxidizing species is formed. This species is stabilized by the negative charge of succinate on the iron. The binding sites of succinate and penicillin overlap, and when penicillin replaces succinate, it removes the stabilizing charge, eliciting oxidative attack on itself. Requisite groups of penicillin are within 1 A of the expected position of a ferryl oxygen in the enzyme-penicillin complex.

Incorporation of oxygen into the succinate co‐product of iron(II) and 2‐oxoglutarate dependent oxygenases from bacteria, plants and humans

“…When ANS catalyses a typical hydroxylation, e.g., the hydroxylation of (±)-naringenin, high levels of incorporation occurred [13]. The deacetoxycephalosporin C synthase (DAOCS, a bacterial enzyme that only catalyses the deacetoxycephalosporin C synthase component of the two reactions of its fungal homologue deacetoxy/deacetylcephalosporin C synthase) reaction might also be regarded as being atypical; there is crystallographic evidence that the oxidative rearrangement catalysed by DAOCS proceeds via a divergence from the more general reaction scheme in which a penicillin displaces succinate from a ferryl intermediate [28]. Clavaminc acid synthase is also unusual in that it catalyses three reactions and has an HXE rather than and HXD Fe(II) binding motif [29].…”

Structural and Mechanistic Studies on the Inhibition of the Hypoxia-inducible Transcription Factor Hydroxylases by Tricarboxylic Acid Cycle Intermediates

“…trans to Asp-201 of FIH or equivalent residue), consistent with labeling and other studies demonstrating that it is derived from the 2-oxo group and dioxygen. One structure of a DAOCS-Fe(II) mutant (⌬R306A) complexed with succinate and carbon dioxide has been reported (PDB code 1E5H) (54) in which the succinate adopts an unusual position, but this complex may be atypical as there is evidence that aspects of the DAOCS mechanism are unusual (9,49). Another exception is in the alkylsulfatase structure (PDB code 1VZ5), in which there is no Fe(II) present for the succinyl carboxylate to coordinate.…”

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

“…Recently, Valegård et al [24 ] have explained how the extremely unstable iron (IV) in DAOCS is kept in check until its reactivity is required. By elucidating several crystal structures of enzyme-substrate and enzyme-product complexes (PDB codes 1UO9, 1UOF, 1UOB, 1UNB and 1UOG), they show that the binding sites for the 2-oxoacid and the b-lactam overlap.…”