Isopenicillin N Synthase Binds δ‐(L‐α‐Aminoadipoyl)‐L‐Cysteinyl‐D‐Thia‐allo‐Isoleucine through both Sulfur Atoms

Abstract

Isopenicillin N synthase (IPNS) catalyses the synthesis of isopenicillin N (IPN), the biosynthetic precursor to penicillin and cephalosporin antibiotics. IPNS is a non-heme iron(II) oxidase that mediates the oxidative cyclisation of the tripeptide δ-L-α-aminoadipoyl-L-cysteinyl-D-valine (ACV) to IPN with a concomitant reduction of molecular oxygen to water. Solution-phase incubation experiments have shown that, although IPNS can turn over analogues with a diverse range of hydrocarbon side chains in the third (valinyl) position of its substrate, the enzyme is much less tolerant of polar residues in this position. Thus, although IPNS converts δ-L-α-aminoadipoyl-L-cysteinyl-D-isoleucine (ACI) and AC-D-allo-isoleucine (ACaI) to penam products, the isosteric sulfur-containing peptides AC-D-thiaisoleucine (ACtI) and AC-D-thia-allo-isoleucine (ACtaI) are not turned over. To determine why these peptides are not substrates, we crystallized ACtaI with IPNS. We report the synthesis of ACtaI and the crystal structure of the IPNS:Fe(II) :ACtaI complex to 1.79 Å resolution. This structure reveals direct ligation of the thioether side chain to iron: the sulfide sulfur sits 2.66 Å from the metal, squarely in the oxygen binding site. This result articulates a structural basis for the failure of IPNS to turn over these substrates.

The Interaction of Isopenicillin N Synthase with Homologated Substrate Analogues δ‐(L‐α‐Aminoadipoyl)‐L‐homocysteinyl‐D‐Xaa Characterised by Protein Crystallography

“…22 In addition, the S ‐methylcysteinyl sulfur also coordinates to iron, as expected. The thioether sulfur sits 2.63 Å from the metal, a distance similar to those seen previously with δ‐( L ‐α‐aminoadipoyl)‐ L ‐cysteinyl‐ D ‐ S ‐methylcysteine (ACmC, 2.69 Å),10 δ‐( L ‐α‐aminoadipoyl)‐ L ‐cysteinyl‐ D ‐thioisoleucine (ACtI, 2.66 Å)32 and δ‐( L ‐α‐aminoadipoyl)‐ L ‐cysteinyl‐ D ‐methionine (ACM, 2.57 Å) 33. As a consequence the metal is hexacoordinate, in contrast to the IPNS:Fe II :ACV and IPNS:Fe II :AmC O V complexes9, 22 and to conformation A of the IPNS:Fe II :AhCV complex.…”

“…It was envisaged that coordination of the S ‐methylcysteinyl sulfur to iron would provide an extra anchor relative to AhCV ( 4 ) because thioether ligation to iron in the IPNS active site is well precedented 10. 25, 32, 33…”

The crystal structure of an isopenicillin N synthase complex with an ethereal substrate analogue reveals water in the oxygen binding site

“…Hexacoordinate iron has previously been observed in IPNS complexes: (i) with analogues that incorporate a methyl sulfide in their third residue (e.g. AC‐ d ‐thia‐ allo ‐isoleucine (ACt a I, 9 ) [20] and AC‐ d ‐methionine [49]), in which the affinity of sulfur for iron means that the sulfide S coordinates to the metal; (ii) with smaller substrate analogues (e.g. AC‐Gly and AC‐ d ‐Ala,[28] AC‐ d ‐α‐aminobutyrate [26], AC‐ d ‐vinylglycine [23] and the dipeptide δ‐ l ‐α‐aminoadipoyl‐ l ‐homocysteine (AhC) [50]), where the smaller side‐chain leaves room for a second water ligand to bind to iron opposite Asp216; and (iii) with lll ‐configured substrates (e.g.…”

“…ACI 6 and AC a I 7 are both cyclized to penicillin products by IPNS, with retention of configuration in the C–S bond‐forming step [18,19]. However neither of the corresponding sulfur‐containing compounds AC‐ d ‐thioisoleucine (ACtI, 8 ) and AC‐ d ‐thio‐ allo ‐isoleucine (ACt a I, 9 ) is turned over by the enzyme [14,20]. It was concluded in 1989 that the difference between the two series ( 3 and 4 on the one hand, 6 and 7 on the other) “must derive from some property of the methoxy function,” and proposed that “a hydrogen bond between the methoxy group and an active site group restricts rotation around C(2)–C(3) in the intermediate … so the [intermediate] derived from the allo ‐threonine peptide can be cyclised, but that derived from the threonine [peptide] is restrained in a conformation in which the β‐hydrogen atom cannot be attacked by the active species” [21].…”

Isopenicillin N Synthase: Crystallographic Studies

“…A) Anaerobic IPNSÀ FeÀ ACtaI complex, in which the methylsulfide of thia-alloisoleucine is ligated in the oxygen binding site (PDB ID: 2Y6F). [29] B) Anaerobic IPNSÀ FeÀ ACM complex, in which the methylsulfide of methionine is similarly ligated in the oxygen binding site (PDB ID: 2Y60). [51] Figure 21.…”