PDBsum entry 1hb4

Antibiotic biosynthesis

PDB id: 1hb4

Contents

Protein chain

329 a.a. *

Ligands

SCV

SO4

Metals

FE2

Waters ×357

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Alternative oxidation by isopenicillin n synthase observed by x-Ray diffraction.

Authors

J.M.Ogle, I.J.Clifton, P.J.Rutledge, J.M.Elkins, N.I.Burzlaff, R.M.Adlington, P.L.Roach, J.E.Baldwin.

Ref.

Chem Biol, 2001, 8, 1231-1237. [DOI no: 10.1016/S1074-5521(01)00090-4]

PubMed id

11755401

Abstract

BACKGROUND: Isopenicillin N synthase (IPNS) catalyses formation of bicyclic isopenicillin N, precursor to all penicillin and cephalosporin antibiotics, from the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine. IPNS is a non-haem iron(II)-dependent enzyme which utilises the full oxidising potential of molecular oxygen in catalysing the bicyclisation reaction. The reaction mechanism is believed to involve initial formation of the beta-lactam ring (via a thioaldehyde intermediate) to give an iron(IV)-oxo species, which then mediates closure of the 5-membered thiazolidine ring. RESULTS: Here we report experiments employing time-resolved crystallography to observe turnover of an isosteric substrate analogue designed to intercept the catalytic pathway at an early stage. Reaction in the crystalline enzyme-substrate complex was initiated by the application of high-pressure oxygen, and subsequent flash freezing allowed an oxygenated product to be trapped, bound at the iron centre. A mechanism for formation of the observed thiocarboxylate product is proposed. CONCLUSIONS: In the absence of its natural reaction partner (the N-H proton of the L-cysteinyl-D-valine amide bond), the proposed hydroperoxide intermediate appears to attack the putative thioaldehyde species directly. These results shed light on the events preceding beta-lactam closure in the IPNS reaction cycle, and enhance our understanding of the mechanism for reaction of the enzyme with its natural substrate.

Figure 1.
Fig. 1. Mechanisms for the reaction of IPNS with ACV and ACOV. (a) Proposed mechanism for the reaction of IPNS with its natural substrate ACV [3]. (b) Proposed pathway for generation of the thiocarboxylate product from the substrate analogue ACOV. See text for details of compounds 1–10. AA, δ-( Image -α-aminoadipoyl).

Figure 4.
Fig. 4. Potential hydrogen bonding interactions in the active site region of the exposed IPNS:Fe(II):ACOV structure. The three exposed structures oriented to show the potential for hydrogen bonding around the thiocarboxylate oxygen, and to demonstrate the consistency of the thiocarboxylate electron density across the three structures. (a) From the 1.30 Å resolution structure in the plane of the thiocarboxylate, showing distances to Wat 402 and Wat 403. (b) The 1.30 Å 30 s, (c) the 1.40 Å 30 s, and (d) the 1.50 Å 120 s structures from an alternative angle.

The above figures are reprinted by permission from Cell Press: Chem Biol (2001, 8, 1231-1237) copyright 2001.

Secondary reference #1

Title

The reaction cycle of isopenicillin n synthase observed by X-Ray diffraction.

Authors

N.I.Burzlaff, P.J.Rutledge, I.J.Clifton, C.M.Hensgens, M.Pickford, R.M.Adlington, P.L.Roach, J.E.Baldwin.

Ref.

Nature, 1999, 401, 721-724. [DOI no: 10.1038/44400]

PubMed id

10537113

Figure 2.
Figure 2 Proposed mechanisms for the oxidation of ACV and ACmC to bicyclic and monocyclic products, respectively. See text for details of compounds 1-6. AA, L- -( -aminoadipoyl).

Figure 3.
Figure 3 Stereo views of the two substrates and two products overlaid. The key regions that participate in the reaction and the iron atom (orange) are shown; the aminoadipoyl side chain, which does not move significantly, is omitted for clarity. Shown are ACV (white), IPN (yellow), ACmC (blue) and its monocyclic sulphoxide product (pink). Figures were prepared using the programs MOLSCRIPT20 and Raster3D (ref. 21).

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd

Secondary reference #2

Title

Structure of isopenicillin n synthase complexed with substrate and the mechanism of penicillin formation.

Authors

P.L.Roach, I.J.Clifton, C.M.Hensgens, N.Shibata, C.J.Schofield, J.Hajdu, J.E.Baldwin.

Ref.

Nature, 1997, 387, 827-830. [DOI no: 10.1038/42990]

PubMed id

9194566

Figure 2.
Figure 2 Mechanism for isopenicillin N formation and the formation of the Fe: ACV: NO:. sp;IPNS complex.

Figure 3.
Figure 3 Comparison of the structures of Mn: IPNS (a) and Fe(II): ACV: IPNS (. b) from the same orientation. The jelly-roll motif is in green, the C-terminal region (residues 313-331) cyan, the active-site metal ion (manganese in a, iron in b) orange, the key substrate-binding residues (His 214, His 270, Asp 216, Arg 87, Arg 279, Tyr 189 and Ser 281) magenta, and the ACV yellow.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd

Secondary reference #3

Title

Crystal structure of isopenicillin n synthase is the first from a new structural family of enzymes.

Authors

P.L.Roach, I.J.Clifton, V.Fülöp, K.Harlos, G.J.Barton, J.Hajdu, I.Andersson, C.J.Schofield, J.E.Baldwin.

Ref.

Nature, 1995, 375, 700-704.

PubMed id

7791906