M-CSA Mechanism and Catalytic Site Atlas

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 Reaction (EC:1.21.3.1)

dioxygen

CHEBI:15379

N-[(5S)-5-ammonio-5-carboxylatopentanoyl]-L-cysteinyl-D-valinate

CHEBI:58572

→

water

CHEBI:15377

isopenicillin N(1-)

CHEBI:58399

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: Isopenicillin N synthase,

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

*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

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 formed

References

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.

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Catalytic 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 used

Step 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.

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Catalytic 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 ion

Step 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.

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Catalytic 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 formation

Step 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

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Catalytic Residues Roles

Residue	Roles
His270A	metal ligand
Asp216A	metal ligand
His214A	metal ligand

Chemical Components

proton transfer, electron transfer

Step 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.

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Catalytic 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, cyclisation

Step 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).

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Catalytic 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 transfer

Step 7. The radical intermediate drives the thiazoladine closure via colligation from the homolysis of the S-Fe bond.

Download: Image, Marvin File

Catalytic 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 formed

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.

Step 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.

Step 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).

Step 7. The radical intermediate drives the thiazoladine closure via colligation from the homolysis of the S-Fe bond.

Products.

Introduction

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 step

References

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 File

Catalytic 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 used

Step 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 File

Catalytic 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 ion

Download: Image, Marvin File

Catalytic 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 formation

Step 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 File

Catalytic 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, cyclisation

Step 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.

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Catalytic 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 formed

Step 6. Inferred return step in which the coordination sphere of the iron cofactor is regenerated by proton transfer from water.

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Catalytic Residues Roles

Residue	Roles
His214A	metal ligand
His270A	metal ligand
Asp216A	metal ligand

Chemical Components

proton transfer, intermediate terminated, native state of enzyme regenerated, native state of cofactor regenerated, inferred reaction step

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.

Step 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.

Step 6. Inferred return step in which the coordination sphere of the iron cofactor is regenerated by proton transfer from water.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Mei Leung, Charity Hornby