Isopenicillin-N synthase

Isopenicillin-N synthase (IPNS) is a non-heme iron-dependent enzyme belonging to the oxidoreductase family. This enzyme catalyzes the formation of isopenicillin-N from δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (LLD-ACV).

:N- [(5S)-5-amino-5-carboxypentanoyl] -L-cysteinyl-D-valine + O₂ $ightleftharpoons$ isopenicillin N + 2 H₂O

This reaction is a key step in the biosynthesis of penicillin and cephalosporin antibiotics [Cohen, G., Shiffman, D., Moshe, M., & Aharonowitz, Y. (1990) Microbial isopenicillin N synthase genes: structure, function, diversity, and evolution. "TIBTECH" 8, 105-111.] . The active site of most isopenicillin-N synthases contains an iron ion [Roach, P.L., Clifton, I.J., Hensgens, C.M.H., Shibata, N., Schofield, C.J., Hajdu J., & Baldwin, J.E. (1997) Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation. "Nature" 387, 827-830.] .

This enzyme is also called isopenicillin N synthetase.

Mechanism

A Fe(II) metal ion in the active site of the enzyme is coordinated by at least two histidine residues, an aspartate residue, a glutamine residue, and two water molecules in the absence of a bound substrate [Roach, P.L., Clifton, I.J., Hensgens, C.M.H., Shibata, N., Schofield, C.J., Hajdu J., & Baldwin, J.E. (1997) Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation. "Nature" 387, 827-830.] . Just two histidine residues and one aspartic acid residue are entirely conserved. Therefore, it is highly significant that these two histidine residues, His214 and His270, and one aspartic acid residue, Asp216, are precisely the ones essential for activity [Borovok, I., Landman, O., Kreisberg-Zakarin, R., Aharonowitz, Y., & Cohen, G. (1996) Ferrous active site of isopenicillin n synthase: genetic and sequence analysis of the endogenous ligands. "Biochemistry" 35, 1981-1987.] . When ACV binds the active site, Gln330 and one water molecule are replaced by the ACV thiolate [Roach, P.L., Clifton, I.J., Hensgens, C.M.H., Shibata, N., Schofield, C.J., Hajdu J., & Baldwin, J.E. (1997) Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation. "Nature" 387, 827-830.] .

The linear tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (LLD-ACV) must first be assembled from its component amino acids by N-(5-amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase (ACV synthase) [Schenk, W.A. (2000) Isopenicillin N synthase: an enzyme at work. "Angew. Chem. Int. Ed." 39, No. 19, 3409-3411.] . This allows for the binding of the substrate ACV to the deprotonated thiol group of the cysteine residue. This ligation of the thiolate to the iron center anchors the ACV within the active site [Roach, P.L., Clifton, I.J., Hensgens, C.M.H., Shibata, N., Schofield, C.J., Hajdu J., & Baldwin, J.E. (1997) Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation. "Nature" 387, 827-830.] .

The ligation of ACV results in a reduction of the FeI/FeII redox potential. This allows dioxygen to bind, which changes the oxidation state, initiating the reaction cycle [Roach, P.L., Clifton, I.J., Hensgens, C.M.H., Shibata, N., Schofield, C.J., Hajdu J., & Baldwin, J.E. (1997) Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation. "Nature" 387, 827-830.] . An intramolecular hydrogen transfer takes place from C-3 of the cysteine residue, taking the iron back to the +II state. A thioaldehyde and a hydroperoxy ligand are produced in this process. The hydroperoxy ligand deprotonates the amide which then closes the β-lactam ring by a nucleophilic attack at the thioaldehyde carbon [Schenk, W.A. (2000) Isopenicillin N synthase: an enzyme at work. "Angew. Chem. Int. Ed." 39, No. 19, 3409-3411.] .

This causes the hydrogen atom at the C-3 of the valine residue to come closer to the iron(IV) oxo ligand which is highly electrophilic. A second hydrogen transfer occurs, most likely producing an isopropyl radical which closes the thiazolidine ring by attacking the thiolate sulfur atom [Schenk, W.A. (2000) Isopenicillin N synthase: an enzyme at work. "Angew. Chem. Int. Ed." 39, No. 19, 3409-3411.] .

Role in Antibiotic Formation

Following the IPNS pathway, further enzymes are responsible for the epimerization of isopenicillin N to penicillin N, the derivitazation to other penicillins, and the ring expansion that eventually leads to the various cephalosporins [Cohen, G., Shiffman, D., Moshe, M., & Aharonowitz, Y. (1990) Microbial isopenicillin N synthase genes: structure, function, diversity, and evolution. "TIBTECH" 8, 105-111.] .

This shows how IPNS occupies an early and key role in the biosynthetic pathway of all of the penicillins and cephalosporins, which are different types of β-lactam antibiotics. This class of antibiotics is the most widely used of any available antibiotic available. They act by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls, which is especially important in Gram-positive organisms.

There are several types of penicillins that can be used to treat different kinds of infections such as skin infections, dental infections, ear infections, respiratory tract infections, urinary tract infections, gonorrhea, and other infections caused by bacteria. They will not work for infections cause by viruses, such as colds or the flu [drug. (2007). In "Encyclopædia Britannica". Retrieved from Encyclopædia Britannica Online: [http://www.britannica.com/eb/article-233923] ] .

tructural studies

As of late 2007, 26 structures have been solved for this class of enzymes, with PDB accession codes PDB link|1ODM, PDB link|1UZW, PDB link|1W03, PDB link|1W04, PDB link|1W05, PDB link|1W06, PDB link|1W3V, PDB link|1W3X, PDB link|2BJS, PDB link|2BU9, PDB link|2IVI, PDB link|2IVJ, [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2jb4 2JB4] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1qje 1QJE] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1ODN 1ODN] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1HB1 1HB1] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1HB2 1HB2] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1HB3 1HB3] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1HB4 1HB4] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1qiq 1QIQ] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1QJF 1QJF] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1bk0 1BK0] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1blz 1BLZ] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1obn 1OBN] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1oc1 1OC1] , [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1ips 1IPS]

References

*
*

External links

Gene Ontology (GO) codes