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

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.

Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in β-lactam biosynthesis

“…5B ). 29 , 57 , 61 Conformational changes at the active site upon binding of ACV are shown in Fig. 5D .…”

“…Recently reported results for a C -terminally truncated IPNS variant support the proposal that the C -terminal α-helix shields the active site and hinders (potential) side reactions during catalysis. 61 …”

Manipulating Gibberellin Control Over Growth and Fertility as a Possible Target for Managing Wild Radish Weed Populations in Cropping Systems

“…Additionally, deletion within the equivalent C-terminal region in C 20 -GA2OXs, eliminates or greatly reduces enzyme activity (Lo et al, 2008). The Cterminal tail also influences specificity and activity in the non-GAOX 2ODDs of DEACETOXYCEPHALOSPORIN C SYNTHASE (DAOCS), ISOPENICILLIN N SYNTHASE (IPNS), and PROLYL HYDROXYLASE DOMAIN 2 (PHD2) (Lee et al, 2001;Chowdhury et al, 2016;McNeill et al, 2017).…”

“…Whereas, a substitution of His(H) > Tyr(Y) at position 276 reduces the GA 3-oxidase efficiency of the pea le-3 mutant allele (Martin et al, 1997) and a Cys(C) > Tyr(Y) at position 219 eliminates GA 3-oxidase activity in the Arabidopsis 3ox1-1 mutant (Chiang et al, 1995). Even residues with similar properties and only minor steric changes [e.g., Leu(L) to Ile(I), Asp(D) to Glu(E), or Lys(K) to Arg(R)] can be poorly tolerated in the 2ODD reaction cavity, leading to altered activity rates or changes in substrate specificity (Gebhardt et al, 2007;McNeill et al, 2017). This is also a factor specifically for GA recognition, where minor steric substitutions in the GA binding pocket of the GID1 GA receptor, lead to increased affinity for either GA 34 or GA 9 at the expense of GA 4 (Shimada et al, 2008).…”

Adventures in Defining Roles of Oxygenases in the Regulation of Protein Biosynthesis

“…There are now multiple examples of the relaxed substrate/product selectivities of 2OG oxygenases, as well as for ACCO and IPNS. The ability of IPNS to convert tripeptide analogues of ACC to multiple bicyclic β‐lactams is particularly striking,,, especially given the relative inaccessibility of many of these product types to non‐enzymatic synthetic chemistry.…”