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Last modified: 1 February 1999

Isopenicillin N synthase (IPNS)
Mononuclear iron centre Iron ligands Formal iron
oxidation/spin states
IPNS Fe centre

Fe(NepsilonHis)2OdeltaAspOepsilonGln·2H2O
2 × NepsilonHis;

OdeltaAsp;

OepsilonGln;

2 × H2O

FeII (S = 2);

FeIV (S = 1)
IPNS Fe-ACV-NO complex

Fe(NepsilonHis)2OdeltaAspSgammaCysNO·H2O
2 × NepsilonHis;

OdeltaAsp;

SgammaCys (substrate);

H2O;

NO or O2

FeII-NO (S = 3/2)

Penicillins and cephalosporins belong to the ß­lactam class of antibiotics. Fungi synthesise either penicillins or cephalosporins, whereas bacteria produce a variety of cephalosporins and cephamycins but do not produce penicillins as end products [1]. All these antibiotics contain a ß­lactam ring fused to a second sulphur­containing ring.

Penicillin is synthesised from three amino acid precursors: L­alpha­aminoadipic acid, L­cysteine and L­valine. Isopenicillin N synthase (IPNS; EC 1.8.99.-) catalyses the oxidative ring closure of the tripeptide delta­(L­alpha­aminoadipoyl)­L­cysteinyl­D­valine (ACV) to form isopenicillin N (1). The enzyme uses one molecule of dioxygen (O2) as the sole electron acceptor, giving two molecules of water; the four electrons required for the O2 reduction originate from ACV [2].

IPNS enzymes are present both in fungi and bacteria and show high sequence similarity. The phylogenetic studies of IPNS gene sequences suggest that the IPNS genes were horizontally transferred from bacterial ß­lactam producers to filamentous fungi [3-5].

Biosynthesis of cephalosporin antibiotics involves an expansion of the five­membered thiazolidine ring of penicillin N to the six­membered dihydrothiazine ring of deacetoxycephalosporin C (DAOC) by a DAOC synthetase (DAOCS or expandase) enzyme activity. The next step in biosynthesis of cephalosporins is the hydroxylation of DAOC to deacetylcephalosporin (DAC) by DAC synthetase (DACS or DAOC hydroxylase) [6]. A bifunctional enzyme deacetoxycephalosporin C synthetase/hydroxylase (DAOCS/DACS) from Cephalosporium acremonium [7] catalyses both of these consecutive reactions. Streptomyces clavuligerus possesses two separate enzymes, DAOCS (expandase) and DACS (hydroxylase) [6, 8]. S. clavuligerus DAOCS shows an extensive sequence similarity to C. acremonium DAOCS/DACS [7]. DACS from Streptomyces clavuligerus, in addition to its main activity, catalyse the hydroxylation of 3­exomethylenecephalosporin C to DAOC and, less effectively, the ring expansion of penicillin N to DAOC; moreover, S. clavuligerus DACS and DAOCS appear to be products of a divergent evolution [8]. DAOCS/DACS, DAOCS and DACS contain FeII as a prosthetic group and share sequence similarity with IPNS.

The 3­D structure of the MnII­substituted IPNS from Aspergillus nidulans has been determined [9]. IPNS is an alpha/ß protein. Metal ion is coordinated by four protein ligands (His­214, His­270, Asp­216 and Gln­330) and two solvent­derived ligands.

A reaction mechanism for IPNS has been proposed [2].

The resting enzyme contains a high­spin, hexacoordinate FeII centre. The initial steps of the reaction are the consecutive binding of the ACV thiolate and O2 which displace one water and the Gln­derived amide ligand to form the ternary complex ACV-FeII-O2 (I). The two­electron oxidation of the Cys thiol of ACV gives a thioaldehyde, while O2 is reduced by two electrons to the peroxide (II). Heterolysis of a peroxo intermediate (II) results in the oxyferryl (ACV-FeIV=O) intermediate (III). A further step may involve recombination of a transient valinyl radical and coordinated thiolate of ACV to yield the thiazolidine ring of isopenicillin N and native enzyme (see [2] for details).

IPNS in motif databases

PRINTS ID PRINTS AC PROSITE/BLOCKS ID PROSITE AC BLOCKS AC
IPNSYNTHASE PR00682 IPNS1
IPNS2		 PS00185
PS00186		 BL00185

IPNS in alignment databases

Protein Superfamily Pfam LPFC 3­D alignment
00088; isopenicillin N synthase
-
-

IPNS in 3­D databases

IPNS contains a single iron atom which has been substituted by manganese in 1IPS (see Figure 1IPS).

PDB scop BSM RELI
Base		 Header MMS Abstract ¹
1bk0
-
 1bk0
-
 Isopenicillin N synthase (ferrous) (complex with delta­(L­alpha­aminoadipoyl)­L­cysteinyl­D­valine and SO42-); Aspergillus nidulans
-
1blz
-
 1blz
-
 Isopenicillin N synthase (ferrous) (complex with NO and delta­(L­alpha­aminoadipoyl)­L­cysteinyl­D­valine); Aspergillus nidulans
-
1ips 1ips 1ips 1ips Isopenicillin N synthase (Mn2+ substituted for Fe2+); Aspergillus nidulans MS6EG5

¹ Macromolecular Structures abstract. Full text is available to BioMedNet Members

References

  1. Aharonowitz, Y., Cohen, G. and Martin, J.F. (1992) Penicillin and cephalosporin biosynthetic genes: Structure, organization, regulation, and evolution. Annu. Rev. Microbiol. 46, 461-495.
  2. Que, L., Jr. and Ho, R.Y.N. (1996) Dioxygen activation by enzymes with mononuclear non­heme iron active sites. Chem. Rev. 96, 2607-2624.
  3. Landan, G., Cohen, G., Aharonowitz, Y., Shuali, Y., Graur, D. and Shiffman, D. (1990) Evolution of isopenicillin N synthase genes may have involved horizontal gene transfer. Mol. Biol. Evol. 7, 399-406.
  4. Penalva, M.A., Moya, A., Dopazo, J. and Ramon, D. (1990) Sequences of isopenicillin N synthetase genes suggest horizontal gene transfer from prokaryotes to eukaryotes. Proc. R. Soc. Lond. B241, 164-169.
  5. Buades, C. and Moya, A. (1996) Phylogenetic analysis of the isopenicillin­N­synthetase horizontal gene transfer. J. Mol. Evol. 42, 537-542.
  6. Kovacevic, S., Weigel, B.J., Tobin, M.B., Ingolia, T.D. and Miller, J.R. (1989) Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase. J. Bacteriol. 171, 754-760.
  7. Dotzlaf, J.E. and Yeh, W.K. (1987) Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium. J. Bacteriol. 169, 1611-1618.
  8. Baker, B.J., Dotzlaf, J.E. and Yeh, W.K. (1991) Deacetoxycephalosporin C hydroxylase of Streptomyces clavuligerus. Purification, characterization, bifunctionality, and evolutionary implication. J. Biol. Chem. 266, 5087-5093.
  9. Roach, P.L., Clifton, I.J., Fülöp, V., Harlos, K., Barton, G.J., Hajdu, J., Andersson, I., Schofield, C.J. and Baldwin, J.E. (1995) Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes. Nature 375, 700-704.
Bibliography on structural studies of isopenicillin N synthase
Reviews on isopenicillin N synthase