Abstract
Isopenicillin N synthase (IPNS) catalyses the four‐electron oxidation of a tripeptide, l‐δ‐(α‐aminoadipoyl)‐l‐cysteinyl‐d‐valine (ACV), to give isopenicillin N (IPN), the first‐formed β‐lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a 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 iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C‐terminal residues, which move to take up a conformation that extends the final α‐helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C‐terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side‐chains around the ACV binding pocket is important in catalysis. 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 an X‐ray crystal structure, 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.
Highlights
Isopenicillin N synthase (IPNS) catalyses the 4-electron oxidation of the tripeptide l-d-(a-aminoadipoyl)-l-cysteinyl-d-valine (ACV, 1) to give bicyclic isopenicillin N (IPN, 2) with concomitant reduction of one molecule of dioxygen to two water molecules (Scheme 1 a).[1]IPNS is part of the superfamily of iron(II) dependent oxidases.[2,3,4] Crystal structures of the Aspergillus nidulans protein[5,6,7] show IPNS to contain the double-stranded b-helix fold (DSBH, known as a “jelly-roll”, cupin or jumonji C (JmjC) fold) characteristic of the 2-oxoglutarate-dependent oxygenase superfamily (Figure S1, Supporting Information).[8]
Product distributions with the “tailless” I325* mutant Previous work with I325* IPNS has shown it retains the ability to convert ACV 1 to IPN 2, but with only 10 % of wildtype activity.[30]
Further examination of elution profiles from HPLC analyses of ACV turnover by the I325* truncated variant revealed a new “split” peak, due to a new product designated as compound 7. This new product eluted from the C18 column between IPN 2 and ACV thiol 1 (Figure S2); the production of 7 increased with time at approximately the same rate as IPN 2, and was not observed in control reactions using heat inactivated enzyme
Summary
Isopenicillin N synthase (IPNS) catalyses the 4-electron oxidation of the tripeptide l-d-(a-aminoadipoyl)-l-cysteinyl-d-valine (ACV, 1) to give bicyclic isopenicillin N (IPN, 2) with concomitant reduction of one molecule of dioxygen to two water molecules (Scheme 1 a).[1]. Turnover of l-d-(a-aminoadipoyl)-l-cysteinyl-glycine (ACG 3, glycine replaces valine) does not generate a bicyclic product; the 5-membered thiazolidine ring cannot form, so the monocyclic b-lactam intermediate opens, giving a 2-electron oxidised shunt product in which the cysteine residue is oxidised to an aldehyde (observed as the hydrate 4) through a two-electron oxidation (Scheme 1 b).[13,19] By contrast, the depsipeptide substrate analogue d-(l-a-aminoadipoyl)-l-cysteine d-a-hydroxyisovaleryl ester (ACOV 5, ester replaces amide between cysteine and valine), cannot form a blactam intermediate.[15] Instead, both oxidising equivalents are directed at the cysteinyl b-carbon, which is converted from the thiol in 5 to the thiocarboxylic acid 6 through 4-electron oxidation (Scheme 1 c) These and other studies, including a range of computational and spectroscopic investigations,[20,21,22,23,24] have led to a detailed chemical understanding of IPNS catalysis, and of the roles played by some key active site residues beyond the iron-binding residues.[25] The side-chain amide carbonyl oxygen of. We report the characterisation of this enzymatically unprecedented product as 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 b-carbon of the other
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