isopenicillin N synthase

{{infobox enzyme

| Name = isopenicillin N synthase

| EC_number = 1.21.3.1

| CAS_number = 78642-31-6

| GO_code = 0016216

| image =

| width =

| caption =

}}

File:IPNS RIBBON.JPG [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1bk0 1BK0] ]]

Isopenicillin N synthase (IPNS) is a non-heme iron protein belonging to the 2-oxoglutarate (2OG)-dependent dioxygenases 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 $\rightleftharpoons$ isopenicillin N + 2 H₂O

This reaction is a key step in the biosynthesis of penicillin and cephalosporin antibiotics.{{cite journal | vauthors = Cohen G, Shiffman D, Mevarech M, Aharonowitz Y | title = Microbial isopenicillin N synthase genes: structure, function, diversity and evolution | journal = Trends Biotechnol. | volume = 8 | issue = 4 | pages = 105–11 |date=April 1990 | pmid = 1366527 | doi = 10.1016/0167-7799(90)90148-Q }}

The active sites of most isopenicillin N synthases contain an iron ion.{{cite journal | vauthors = Roach PL, Clifton IJ, Hensgens CM, Shibata N, Schofield CJ, Hajdu J, Baldwin JE | title = Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation | journal = Nature | volume = 387 | issue = 6635 | pages = 827–30 |date=June 1997 | pmid = 9194566 | doi = 10.1038/42990 | s2cid = 205032251 | doi-access = free | bibcode = 1997Natur.387..827R }}

This enzyme is also called isopenicillin N synthetase.

Mechanism

File:ACTIVE SITE Isopenicillin-N synthase.JPG [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1bk0 1BK0] ]]

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. 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.{{cite journal | vauthors = Borovok I, Landman O, Kreisberg-Zakarin R, Aharonowitz Y, Cohen G | title = Ferrous active site of isopenicillin N synthase: genetic and sequence analysis of the endogenous ligands | journal = Biochemistry | volume = 35 | issue = 6 | pages = 1981–7 |date=February 1996 | pmid = 8639682 | doi = 10.1021/bi951534t }} When ACV binds the active site, Gln330 and one water molecule are replaced by the ACV thiolate.

File:IPNS active site1.gif

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).{{cite journal | vauthors = Schenk WA | title = Isopenicillin N Synthase: An Enzyme at Work | journal = Angew. Chem. Int. Ed. Engl. | volume = 39 | issue = 19 | pages = 3409–3411 | date = October 2000 | pmid = 11091371 | doi = 10.1002/1521-3773(20001002)39:19<3409::AID-ANIE3409>3.0.CO;2-T | doi-access = free | bibcode = 2000AngCh..39.3409S }} 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.

The ligation of ACV results in a decrease in the Fe(II)/Fe(III) redox potential, which facilitates the reduction of dioxygen to superoxide. Formation of a highly covalent RS-Fe(III) bond{{cite journal | vauthors = Kovacs JA, Brines, LM | title = Understanding How the Thiolate Sulfur Contributes to the Function of the Non-Heme Iron Enzyme Superoxide Reductase | journal = Acc. Chem. Res. | volume = 40 | issue = 7 | pages = 501–509 |date=July 2007 | pmid = 17536780 | doi = 10.1021/ar600059h| pmc = 3703784 }}{{cite journal | vauthors = Kovacs JA, Brines, LM | title = VTVH-MCD and DFT Studies of Thiolate Bonding to {FeNO}7/{FeO2}8 Complexes of Isopenicillin N Synthase: Substrate Determination of Oxidase versus Oxygenase Activity in Nonheme Fe Enzymes | journal = J. Am. Chem. Soc. | volume = 129 | issue = 23 | pages = 7427–7438 |date=May 2007 | pmid = 17506560 | doi = 10.1021/ja071364v| pmc = 2536647 }} lowers the activation barrier to dioxygen binding, thereby initiating the reaction cycle. An intramolecular hydrogen atom transfer from C-3 of the cysteine residue (BDE= 93 kcal/mol) to the distal superoxo oxygen takes place, thereby converting the Fe(III) back to the Fe(II) oxidation state. A thioaldehyde and a hydroperoxy ligand (OOH) 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.

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.

File:IPNS mechanism.gif

Role in Antibiotic Formation

File:Antibiotic formation pathway.svg

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.

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. 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 bacterial infections.{{cite book | vauthors = Geddes AM, Williams JD | title = Pharmacology of Antibiotics | date = 1976 | publisher = Springer US | location = Boston, MA | isbn = 978-1-4684-3123-0 }} They will not work for infections caused by viruses, such as colds or the flu.{{cite web | vauthors = Anderson L | title = Why Don't Antibiotics Kill Viruses? | url = https://www.drugs.com/article/antibiotics-and-viruses.html | work = Drugs.com }}

Structural 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]

isopenicillin N synthase

Mechanism

Role in Antibiotic Formation

Structural studies

References

Further reading