Deacetoxycephalosporin-C synthase

 

Deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus is able to catalyse the synthesis of sporins from penicillin using 2-oxoglutarate (2OG) as a co-substrate. This reaction is potentially very useful to industry because it enables processing of penicillin to other beta-lactam ring containing compounds which may not trigger antibiotic resistance systems in prokaryotes that evolved with penicillin itself as the selective agent. As with the other members of the family of 2OG utilising enzymes, DAOCS shows a beta barrel catalytic core and has its iron centre ligated to two histidines and an aspartate residue at the active site. It also displays roughly the same catalytic cycle, albeit for a specific reaction, as seen for the other members of the family, such as anthocyanidin synthase.

 

Reference Protein and Structure

Sequence
P18548 UniProt (1.14.20.1) IPR027443 (Sequence Homologues) (PDB Homologues)
Biological species
Streptomyces clavuligerus (Bacteria) Uniprot
PDB
1unb - Deacetoxycephalosporin C synthase complexed with 2-oxoglutarate and ampicillin (1.5 Å) PDBe PDBsum 1unb
Catalytic CATH Domains
2.60.120.330 CATHdb (see all for 1unb)
Cofactors
Iron(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.14.20.1)

dioxygen
CHEBI:15379ChEBI
+
penicillin N(1-)
CHEBI:58408ChEBI
+
2-oxoglutarate(2-)
CHEBI:16810ChEBI
→
carbon dioxide
CHEBI:16526ChEBI
+
deacetoxycephalosporin C(1-)
CHEBI:58415ChEBI
+
succinate(2-)
CHEBI:30031ChEBI
+
water
CHEBI:15377ChEBI
Alternative enzyme names: DAOCS, Penicillin N expandase, DAOC synthase,

Enzyme Mechanism

Introduction

There are two half reactions that occur in the enzyme which are independent in as much as they can be uncoupled under certain conditions. In the oxidative half reaction the Fe (II) centre binds to dioxygen, acting as a nucleophile thus becoming oxidised to Fe (III). This creates a peroxide radical which reacts with the 2OG bound to the iron causing decarboxylation and the further oxidation of iron to the highly reactive Fe (IV) species with a double bond to oxygen. In the reductive half reaction the double bonded oxygen accepts a hydrogen atom from the beta methyl group of penicillin to form an OH group liganded to the iron (now Fe (III)) and an alkyl radical. The alkyl radical reacts with the sulphur of the ring to cause the expansion of the ring and the migration of the radical electron to the 2C of the ring. From here, hydrogen transfer to the OH group via the Sulphur atom occurs leading to the formation of a double bond between the 2C and the 3C of the ring, and the formation of Fe (II) bonded to a water molecule. This completes the reaction cycle. Arg 74 ensures that the hydrogen transfer can occur by sterically constraining penicillin so that it is in the same plane as the Fe (IV) double bonded oxygen.

Catalytic Residues Roles

UniProt PDB* (1unb)
Arg74 Arg74A Sterically constrains the penicillin substrate thus positioning it in the optimal position for hydrogen transfer. steric hindrance
*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

bimolecular electrophilic addition, coordination, overall reactant used, coordination to a metal ion, intermediate formation, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, decarboxylation, hydrogen transfer, radical formation, redox reaction, colligation, radical propagation, cyclisation, homolysis, decyclisation, electron transfer, proton transfer, radical termination, heterolysis, decoordination from a metal ion, intermediate terminated, native state of enzyme regenerated

References

  1. Clifton IJ et al. (2006), J Inorg Biochem, 100, 644-669. Structural studies on 2-oxoglutarate oxygenases and related double-stranded β-helix fold proteins. DOI:10.1016/j.jinorgbio.2006.01.024. PMID:16513174.
  2. Valegård K et al. (2004), Nat Struct Mol Biol, 11, 95-101. The structural basis of cephalosporin formation in a mononuclear ferrous enzyme. DOI:10.1038/nsmb712. PMID:14718929.
  3. Oster LM et al. (2004), J Mol Biol, 343, 157-171. Conformational Flexibility of the C Terminus with Implications for Substrate Binding and Catalysis Revealed in a New Crystal Form of Deacetoxycephalosporin C Synthase. DOI:10.1016/j.jmb.2004.07.049. PMID:15381427.
  4. Lee HJ et al. (2001), J Mol Biol, 308, 937-948. Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS). DOI:10.1006/jmbi.2001.4649. PMID:11352583.

Step 1. 2-oxoglutarate initiates an electrophilic addition to dioxygen, which coordinates to the Fe(II) cofactor.

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Catalytic Residues Roles

Residue Roles
Arg74A steric hindrance

Chemical Components

ingold: bimolecular electrophilic addition, coordination, overall reactant used, coordination to a metal ion, intermediate formation

Step 2. Carbon dioxide is eliminated from the intermediate to form a planar peroxo intermediate. Carbon dioxide is still coordinated to the Fe(II) cofactor.

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Catalytic Residues Roles

Residue Roles
Arg74A steric hindrance

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse, intermediate formation, overall product formed, decarboxylation

Step 3. Fe(II) initiates an elimination reaction by donating two electrons to the bound peroxo moiety, cleaving the peroxo bond and producing the succinate product and iron as a ferryl species.

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Catalytic Residues Roles

Residue Roles
Arg74A steric hindrance

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate formation, overall product formed

Step 4. Penicillin binds, displacing the succinate and carbon dioxide as iron ligands. The oxo group deprotonates the CH3 of the penicillin, generating a alkyl radical and resulting in the donation of a single electron to Fe(IV).

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Catalytic Residues Roles

Residue Roles
Arg74A steric hindrance

Chemical Components

hydrogen transfer, radical formation, redox reaction, overall reactant used, intermediate formation

Step 5. A colligation occurs between the alkyl radical and the sulfur of the five membered ring in the penicillin.

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Catalytic Residues Roles

Residue Roles
Arg74A steric hindrance

Chemical Components

colligation, radical propagation, intermediate formation, cyclisation

Step 6. The C-S bond of the original five-mebered ring homolyses, producing the six-membered ring.

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Catalytic Residues Roles

Residue Roles
Arg74A steric hindrance

Chemical Components

homolysis, radical propagation, intermediate formation, decyclisation

Step 7. The carbon radical in the ring donates a single electron to the Fe(III) cofactor, which causes the bound hydroxide to deprotonate the C3 of the thiazolidine ring generating the final deacetoxycephalosporin C product.

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Catalytic Residues Roles

Residue Roles
Arg74A steric hindrance

Chemical Components

electron transfer, proton transfer, radical termination, heterolysis, decoordination from a metal ion, intermediate terminated, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. 2-oxoglutarate initiates an electrophilic addition to dioxygen, which coordinates to the Fe(II) cofactor.

Step 2. Carbon dioxide is eliminated from the intermediate to form a planar peroxo intermediate. Carbon dioxide is still coordinated to the Fe(II) cofactor.

Step 3. Fe(II) initiates an elimination reaction by donating two electrons to the bound peroxo moiety, cleaving the peroxo bond and producing the succinate product and iron as a ferryl species.

Step 4. Penicillin binds, displacing the succinate and carbon dioxide as iron ligands. The oxo group deprotonates the CH3 of the penicillin, generating a alkyl radical and resulting in the donation of a single electron to Fe(IV).

Step 5. A colligation occurs between the alkyl radical and the sulfur of the five membered ring in the penicillin.

Step 6. The C-S bond of the original five-mebered ring homolyses, producing the six-membered ring.

Step 7. The carbon radical in the ring donates a single electron to the Fe(III) cofactor, which causes the bound hydroxide to deprotonate the C3 of the thiazolidine ring generating the final deacetoxycephalosporin C product.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Peter Sarkies