Terminally truncated isopenicillin N synthase generates a dithioester product: evidence for a thioaldehyde intermediate during catalysis and a new mode of reaction for non-heme iron oxidases

Isopenicillin N synthase (IPNS) catalyses the oxidation of a tripeptide, L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV), to isopenicillin N (IPN), the first-formed β-lactam in penicillin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as co-substrate. In the absence of substrate, the carbonyl oxygen of the side-chain amide of the penultimate residue, Gln330, co-ordinates to the active site metal. Substrate binding ablates this interaction, triggering rearrangement of seven C-terminal residues which move to take up a conformation that extends the final α-helix and encloses the active site. We report mutagenesis studies probing the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. Unexpectedly, deletion of seven C-terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC-MS and NMR analyses to be the ene-thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl β-carbon of the other. A mechanism for its formation is proposed, supported by X-ray crystal data which shows the substrate ACV bound at the active site, its cysteinyl β-carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non-heme iron oxidases in general

Terminally truncated isopenicillin N synthase generates a dithioester product: evidence for a thioaldehyde intermediate during catalysis and a new mode of reaction for non-heme iron oxidases

Isopenicillin N synthase (IPNS) catalyses the oxidation of a tripeptide, L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV), to isopenicillin N (IPN), the first-formed β-lactam in penicillin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as co-substrate. In the absence of substrate, the carbonyl oxygen of the side-chain amide of the penultimate residue, Gln330, co-ordinates to the active site metal. Substrate binding ablates this interaction, triggering rearrangement of seven C-terminal residues which move to take up a conformation that extends the final α-helix and encloses the active site. We report mutagenesis studies probing the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. Unexpectedly, deletion of seven C-terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC-MS and NMR analyses to be the ene-thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl β-carbon of the other. A mechanism for its formation is proposed, supported by X-ray crystal data which shows the substrate ACV bound at the active site, its cysteinyl β-carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non-heme iron oxidases in general

Regio- and stereodivergent antibiotic oxidative carbocyclizations catalysed by Rieske oxygenase-like enzymes

Oxidative cyclizations, exemplified by the biosynthetic assembly of the penicillin nucleus from a tripeptide precursor, are arguably the most synthetically powerful implementation of C–H activation reactions in nature. Here, we show that Rieske oxygenase-like enzymes mediate regio- and stereodivergent oxidative cyclizations to form 10- and 12-membered carbocyclic rings in the key steps of the biosynthesis of the antibiotics streptorubin B and metacycloprodigiosin, respectively. These reactions represent the first examples of oxidative carbocyclizations catalysed by non-haem iron-dependent oxidases and define a novel type of catalytic activity for Rieske enzymes. A better understanding of how these enzymes achieve such remarkable regio- and stereocontrol in the functionalization of unactivated hydrocarbon chains will greatly facilitate the development of selective man-made C–H activation catalysts

The structural basis of cephalosporin formation in a mononuclear ferrous enzyme

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 Å of the expected position of a ferryl oxygen in the enzyme–penicillin complex.