Isopenicillin-N synthase
Isopenicillin N synthase(IPNS), a non-haem iron-dependent oxidase, catalyses the reaction of delta-(L-aminoadipoyl)-L-cysteinyl-D-valine (ACV) and dioxygen to form isopenicillin N (IPN) and 2 water molecules. IPN is the precursor of the antibiotics, penicillins and cephalosporins.
Reference Protein and Structure
- Sequence
- P05326 (1.21.3.1) (Sequence Homologues) (PDB Homologues)
- Biological species
-
Aspergillus nidulans FGSC A4 (Fungus)
- PDB
- 1odm - ISOPENICILLIN N SYNTHASE FROM ASPERGILLUS NIDULANS (ANAEROBIC AC-VINYLGLYCINE FE COMPLEX) (1.15 Å)
- Catalytic CATH Domains
- 2.60.120.330 (see all for 1odm)
- Cofactors
- Iron(2+) (1) Metal MACiE
Enzyme Mechanism
Introduction
The thio group of ACV cysteinyl residue ligands to the iron and results in a reduction of the Fe(II)/Fe(III) redox potential, allowing dioxygen to bind at the site trans to Asp126, thereby initiating the reaction cycle. With a nonlinear, haemoglobin-like binding of dioxygen, a superoxide is formed, juxtaposed to the pro-3-S hydrogen of the ACV cysteinyl residue, and it abstracts this hydrogen atom to form the thioaldehyde intermediate. Subsequent cleavage of the hydroperoxide along with the deprotonation of the adjacent Fe coordinated water results in the release of a water molecule. This water molecule is then deprotonated by the hydroxide it just deprotonated which enables it to accept a proton from the amide of the intermediate which results in the closure of the beta-lactam ring. After that a homolytic addition occurs between the Fe coordinated oxygen and the tertiary carbon on the substrate which generates a radical intermediate. This radical intermediate can then initiate the thiazoladine closure after the homolysis of the S-Fe bond.
Catalytic Residues Roles
UniProt | PDB* (1odm) | ||
His270, His214, Asp216 | His270A, His214A, Asp216A | Forms Fe binding site | metal ligand |
Chemical Components
proton transfer, bimolecular nucleophilic substitution, overall reactant used, coordination to a metal ion, decoordination from a metal ion, intermediate formation, cofactor used, redox reaction, radical formation, bimolecular homolytic addition, unimolecular elimination by the conjugate base, hydride transfer, coordination, electron transfer, radical termination, intramolecular nucleophilic addition, cyclisation, homolysis, unimolecular homolytic elimination, colligation, enzyme-substrate complex cleavage, overall product formedReferences
- McNeill LA et al. (2017), Chemistry, 23, 12815-12824. Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non-Heme Iron Oxidases. DOI:10.1002/chem.201701592. PMID:28703303.
- Lundberg M et al. (2007), J Phys Chem B, 111, 9380-9389. Protein environment facilitates O2 binding in non-heme iron enzyme. An insight from ONIOM calculations on isopenicillin N synthase (IPNS). DOI:10.1021/jp071878g. PMID:17637052.
Step 1. Thiol of the substrate, delta(L-2-aminoadipyl)-L-cysteinyl-D-valine, coordinates to the iron cofactor in a nucleophilic substitution reaction, eliminating water.
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
Asp216A | metal ligand |
His270A | metal ligand |
His214A | metal ligand |
Chemical Components
proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, coordination to a metal ion, decoordination from a metal ion, intermediate formation, cofactor usedStep 2. The iron cofactor initiates a homolytic addition to dioxygen, resulting in an iron bound dioxygen radical and iron in the Fe(III) oxidation step.
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
Asp216A | metal ligand |
His270A | metal ligand |
His214A | metal ligand |
Chemical Components
redox reaction, radical formation, ingold: bimolecular homolytic addition, overall reactant used, intermediate formation, coordination to a metal ionStep 3. The iron coordinated sulfur eliminates a hydride from the bound intermediate, resulting in a peroxo species bound to the iron, and a single electron transfer back to the iron, regenerating the Fe(II) oxidation state.
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
Asp216A | metal ligand |
His270A | metal ligand |
His214A | metal ligand |
Chemical Components
ingold: unimolecular elimination by the conjugate base, hydride transfer, coordination, electron transfer, radical termination, intermediate formationStep 4. Iron is oxidised from Fe(II) to Fe(IV), forming an oxo group bound to the iron and causing the cleavage of the peroxo O-O bond, which deprotonates the a coordinated water in the Fe coordination sphere
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
His270A | metal ligand |
Asp216A | metal ligand |
His214A | metal ligand |
Chemical Components
proton transfer, electron transferStep 5. The water molecule produced in the last step is deprotonated by the Fe coordinated hydroxyl which enable it to accept a proton from the amide of the intermediate, resulting in the formation of the four-membered beta-lactam ring bound to the iron cofactor.
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
His270A | metal ligand |
Asp216A | metal ligand |
His214A | metal ligand |
Chemical Components
ingold: unimolecular elimination by the conjugate base, ingold: intramolecular nucleophilic addition, proton transfer, intermediate formation, cyclisationStep 6. The oxygen coordinated to Fe initiates a homolytic addition and accepts the proton from the tertiary carbon, resulting in a tertiary carbon radical and converts Fe(IV) to Fe(III).
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
His270A | metal ligand |
Asp216A | metal ligand |
His214A | metal ligand |
Chemical Components
ingold: bimolecular homolytic addition, homolysis, radical formation, intermediate formation, proton transferStep 7. The radical intermediate drives the thiazoladine closure via colligation from the homolysis of the S-Fe bond.
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
His270A | metal ligand |
Asp216A | metal ligand |
His214A | metal ligand |
Chemical Components
ingold: unimolecular homolytic elimination, colligation, radical termination, decoordination from a metal ion, enzyme-substrate complex cleavage, homolysis, overall product formedIntroduction
A mechanism is proposed involving the formation of a monocyclic beta-lactam intermediate. The thio group of ACV cysteinyl residue ligands to the iron and results in a reduction of the Fe(II)/Fe(III) redox potential, allowing dioxygen to bind at the site trans to Asp126, thereby initiating the reaction cycle. With a nonlinear, haemoglobin-like binding of dioxygen, a superoxide is formed, juxtaposed to the pro-3-S hydrogen of the ACV cysteinyl residue, and it abstracts this hydrogen atom to form the thioaldehyde intermediate. Subsequent cleavage of the hydroperoxide with concomitant deprotonation of the amide NH allows simultaneous delta-lactam closure and ferryl formation, yielding one water molecule. Isopropyl group rotates before or at the same time as delta-lactam formation to relieve its steric interactions with the sulphur ligand. This rotation directs the valine beta-hydrogen towards the ferryl-oxo species. The ferryl oxygen deprotonates the valine beta-carbon, resulting in the formation of the thiazolidine ring. Based on the structure, Phe211 may help to isolate the hydroperoxide intermediate from potential hydrogen-bonding partners, enhancing the basicity of the hydroperoxide-hydroxide and facilitating deprotonation of the ACV valine NH.
Catalytic Residues Roles
UniProt | PDB* (1odm) | ||
Phe211 | Phe211A | It isolates the hydroperoxide intermediate from potential hydrogen-bonding partners, enhancing the basicity of the hydroperoxide-hydroxide and facilitating deprotonation of the ACV valine NH. | steric role, polar/non-polar interaction |
His270, His214, Asp216 | His270A, His214A, Asp216A | Forms part of the iron binding site. | metal ligand |
Chemical Components
proton transfer, bimolecular nucleophilic substitution, overall reactant used, coordination to a metal ion, decoordination from a metal ion, intermediate formation, cofactor used, redox reaction, radical formation, bimolecular homolytic addition, unimolecular elimination by the conjugate base, hydride transfer, coordination, electron transfer, radical termination, intramolecular nucleophilic addition, intermediate collapse, overall product formed, cyclisation, intramolecular nucleophilic substitution, intermediate terminated, native state of enzyme regenerated, native state of cofactor regenerated, inferred reaction stepReferences
- Roach PL et al. (1997), Nature, 387, 827-830. Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation. DOI:10.1038/42990. PMID:9194566.
- Burzlaff NI et al. (1999), Nature, 401, 721-724. The reaction cycle of isopenicillin N synthase observed by X-ray diffraction. DOI:10.1038/44400. PMID:10537113.
Step 1. Thiol of the substrate, delta(L-2-aminoadipyl)-L-cysteinyl-D-valine, coordinates to the iron cofactor in a nucleophilic substitution reaction, eliminating water.
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
His214A | metal ligand |
His270A | metal ligand |
Asp216A | metal ligand |
Chemical Components
proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, coordination to a metal ion, decoordination from a metal ion, intermediate formation, cofactor usedStep 2. The iron cofactor initiates a homolytic addition to dioxygen, resulting in an iron bound dioxygen radical and iron in the Fe(III) oxidation step.
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
Phe211A | polar/non-polar interaction |
His214A | metal ligand |
His270A | metal ligand |
Asp216A | metal ligand |
Chemical Components
redox reaction, radical formation, ingold: bimolecular homolytic addition, overall reactant used, intermediate formation, coordination to a metal ionStep 3. The iron coordinated sulfur eliminates a hydride from the bound intermediate, resulting in a peroxo species bound to the iron, and a single electron transfer back to the iron, regenerating the Fe(II) oxidation state.
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
Phe211A | polar/non-polar interaction |
His214A | metal ligand |
His270A | metal ligand |
Asp216A | metal ligand |
Chemical Components
ingold: unimolecular elimination by the conjugate base, hydride transfer, coordination, electron transfer, radical termination, intermediate formationStep 4. Iron is oxidised from Fe(II) to Fe(IV), forming an oxo group bound to the iron and causing the cleavage of the peroxo O-O bond, which deprotonates the amide in the intermediate, resulting in the formation of the four-membered beta-lactam ring bound to the iron cofactor.
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
Phe211A | polar/non-polar interaction, steric role |
His214A | metal ligand |
His270A | metal ligand |
Asp216A | metal ligand |
Chemical Components
ingold: unimolecular elimination by the conjugate base, proton transfer, ingold: intramolecular nucleophilic addition, intermediate collapse, intermediate formation, overall product formed, cyclisationStep 5. The iron-bound sulfur attacks the intermediate in an internal nucleophilic substitution, resulting in the elimination of a hydride ion that attacks the peroxo group, forming a hydride and transferring two electrons back to the iron, regenerating the Fe(II) oxidation state and the isopenicillin N product.
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
His214A | metal ligand |
His270A | metal ligand |
Asp216A | metal ligand |
Chemical Components
proton transfer, ingold: intramolecular nucleophilic substitution, decoordination from a metal ion, intermediate collapse, intermediate formation, overall product formedStep 6. Inferred return step in which the coordination sphere of the iron cofactor is regenerated by proton transfer from water.
Download: Image, Marvin FileCatalytic Residues Roles
Residue | Roles |
---|---|
His214A | metal ligand |
His270A | metal ligand |
Asp216A | metal ligand |