6-deoxyerythronolide B hydroxylase

 

6-deoxyerythronolide B hydroxylase (P450eryF) is a heme-thiolate protein (P-450). P-450s are a superfamily of heme proteins found in all eukaryotes, most prokaryotes, and Archaea and catalyse the monooxygenation of a wide variety of organic molecules. P450 reactions of biological significance include steroid biogenesis, drug metabolism, procarcinogen activation, xenobiotic detoxification, and fatty acid metabolism. P450eryF catalyses the NADPH-dependent conversion of 6-deoxyerythronolide B (6-DEB) to erythronolide B (EB) by the insertion of an oxygen at the 6S position of 6-DEB. Function requires the participation of a ferredoxin and a ferredoxin reductase for the transfer of electrons from NADPH to the monooxygenase.

 

Reference Protein and Structure

Sequence
Q00441 UniProt (1.14.15.35) IPR002397 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharopolyspora erythraea NRRL 2338 (Bacteria) Uniprot
PDB
1oxa - CYTOCHROME P450 (DONOR:O2 OXIDOREDUCTASE) (2.1 Å) PDBe PDBsum 1oxa
Catalytic CATH Domains
1.10.630.10 CATHdb (see all for 1oxa)
Cofactors
Heme b (1)
Click To Show Structure

Enzyme Reaction (EC:1.14.15.35)

hydrogen donor
CHEBI:17499ChEBI
+
6-deoxyerythronolide B
CHEBI:16089ChEBI
+
dioxygen
CHEBI:15379ChEBI
→
erythronolide B
CHEBI:27977ChEBI
+
water
CHEBI:15377ChEBI
+
acceptor
CHEBI:15339ChEBI
Alternative enzyme names: DEB hydroxylase, EryF (gene name), P450(eryF), CYP107A1,

Enzyme Mechanism

Introduction

The catalytic cycle of all cytochrome P450s is conserved. Electrons are delivered to the haem centre from NADH, permitting oxygen reduction. This is illustrated as a general electron donor. Binding of the substrate promotes electron transfer to P450 haem, reducing the iron from Fe(III) to Fe(II). Dioxygen binds and thus Fe(II) is oxidised back to Fe(III). A second electron is passed to the haem forming a superoxide species, followed by donation of two protons. Catalytic water (Wat564), and uniquely the C5 hydroxyl group of 6-DEB, provide the protons to the distil oxygen. This results in the loss of water and the formation of the low spin Fe(IV) oxo complex. Reprotonation of the catalytic water and 6-DEB after protonation of dioxygen occurs via a hydrogen bonded network presumed to be in contact with the bulk solvent during the course of the reaction. The rebound mechanism forms the alcohol group on the substrate once the oxo group is formed. The Fe(IV)-oxo complex undergoes spin inversion to form a radical oxo group which removes a hydrogen from the substrate to form a radical carbon centre and an alcohol group on Fe(IV). The new radical then attacks the oxygen to form the alcohol group and Fe(III), completing the catalytic cycle.

Catalytic Residues Roles

UniProt PDB* (1oxa)
Ala241 (main-C) Ala241(240)A (main-C) Involved in a hydrogen bond network with Glu360, Ser246 and a water molecule which is responsible for protonating the peroxy-complex intermediate. hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Cys351 Cys351(350)A Coordinated to the Fe ion in heme b. metal ligand
Ser246 Ser246(245)A Member of the hydrogen bonded network which replenishes the protons in the active site. hydrogen bond acceptor, hydrogen bond donor, metal ligand, proton acceptor, proton donor, proton relay, electrostatic stabiliser
Glu360 Glu360(359)A Member of the hydrogen bonded network which replenishes the protons in the active site. hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser, proton donor
*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

redox reaction, decoordination from a metal ion, coordination to a metal ion, electron transfer, proton transfer, proton relay, intermediate formation, heterolysis, radical formation, overall reactant used, overall product formed, radical termination, aromatic bimolecular nucleophilic addition

References

  1. Nagano S et al. (2005), J Biol Chem, 280, 22102-22107. Crystal Structures of the Ferrous Dioxygen Complex of Wild-type Cytochrome P450eryF and Its Mutants, A245S and A245T: INVESTIGATION OF THE PROTON TRANSFER SYSTEM IN P450eryF. DOI:10.1074/jbc.m501732200. PMID:15824115.
  2. Sen K et al. (2014), J Phys Chem B, 118, 2810-2820. Role of Two Alternate Water Networks in Compound I Formation in P450eryF. DOI:10.1021/jp411272h. PMID:24564366.
  3. Muralidhara BK et al. (2007), J Am Chem Soc, 129, 2015-2024. Dissecting the Thermodynamics and Cooperativity of Ligand Binding in Cytochrome P450eryF. DOI:10.1021/ja066303w. PMID:17256854.
  4. Choonkeun K et al. (2000), Bioorg Chem, 28, 306-314. The Role of Serine-246 in Cytochrome P450eryF-Catalyzed Hydroxylation of 6-Deoxyerythronolide B. DOI:10.1006/bioo.2000.1187. PMID:11133149.
  5. Cupp-Vickery JR et al. (1996), Nat Struct Biol, 3, 632-637. Substrate-assisted catalysis in cytochrome P450eryF. DOI:10.1038/nsb0796-632. PMID:8673608.
  6. Cupp-Vickery JR et al. (1995), Nat Struct Biol, 2, 144-153. Structure of cytochrome P450eryF involved in erythromycin biosynthesis. PMID:7749919.

Step 1. After the substrate binds a water molecule is released from the active site which increases the redox potential. This facilitates the first one electron reduction of Fe(III) to Fe(II) by NADPH (illustrated here as transfer from a general donor).

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand
Ala241(240)A (main-C) electrostatic stabiliser
Ser246(245)A electrostatic stabiliser
Ala241(240)A (main-C) hydrogen bond donor
Ser246(245)A hydrogen bond donor
Ala241(240)A (main-C) hydrogen bond acceptor
Ser246(245)A hydrogen bond acceptor
Glu360(359)A electrostatic stabiliser, hydrogen bond acceptor, hydrogen bond donor

Chemical Components

redox reaction, decoordination from a metal ion

Step 2. Molecular oxygen binds to Fe(II) to form the oxyferrous intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand
Ala241(240)A (main-C) electrostatic stabiliser
Ser246(245)A electrostatic stabiliser
Glu360(359)A electrostatic stabiliser
Ala241(240)A (main-C) hydrogen bond acceptor
Ser246(245)A hydrogen bond acceptor
Glu360(359)A hydrogen bond acceptor
Ala241(240)A (main-C) hydrogen bond donor
Ser246(245)A hydrogen bond donor
Glu360(359)A hydrogen bond donor

Chemical Components

coordination to a metal ion, redox reaction

Step 3. The second reduction forms a peroxo intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand
Ala241(240)A (main-C) electrostatic stabiliser
Ser246(245)A electrostatic stabiliser
Glu360(359)A electrostatic stabiliser
Ala241(240)A (main-C) hydrogen bond donor
Ser246(245)A hydrogen bond donor
Glu360(359)A hydrogen bond donor
Ala241(240)A (main-C) hydrogen bond acceptor
Ser246(245)A hydrogen bond acceptor
Glu360(359)A hydrogen bond acceptor

Chemical Components

redox reaction, electron transfer

Step 4. The peroxy oxygen accepts a proton from the C5 hydroxyl group of 6-DEB.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala241(240)A (main-C) electrostatic stabiliser
Ser246(245)A electrostatic stabiliser
Glu360(359)A electrostatic stabiliser
Ala241(240)A (main-C) hydrogen bond acceptor
Ser246(245)A hydrogen bond acceptor
Glu360(359)A hydrogen bond acceptor
Ala241(240)A (main-C) hydrogen bond donor
Ser246(245)A hydrogen bond donor
Glu360(359)A hydrogen bond donor
Ser246(245)A metal ligand

Chemical Components

proton transfer

Step 5. A second proton transfer is facilitated by a hydrogen bond network involving Ala241, Ser246 and Glu360.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand
Ser246(245)A proton relay, proton donor
Glu360(359)A proton donor
Ser246(245)A proton acceptor

Chemical Components

proton transfer, proton relay, intermediate formation

Step 6. There is fission of the O-O bond and loss of a water molecule. The proton abstraction from 5-OH by O2 transiently forms a substrate alkoxide. After O–O bond fission, the C5 alkoxide is reprotonated, most likely by the solvent.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand
Ala241(240)A (main-C) electrostatic stabiliser
Ser246(245)A electrostatic stabiliser
Glu360(359)A electrostatic stabiliser
Ala241(240)A (main-C) hydrogen bond acceptor
Ser246(245)A hydrogen bond acceptor
Glu360(359)A hydrogen bond acceptor
Ala241(240)A (main-C) hydrogen bond donor
Ser246(245)A hydrogen bond donor
Glu360(359)A hydrogen bond donor

Chemical Components

heterolysis, proton transfer, proton relay

Step 7. The Fe(IV)-oxo complex undergoes spin inversion to form a radical oxo group which removes a hydrogen from the substrate to form a radical carbon centre and an alcohol group on Fe(IV).

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand

Chemical Components

radical formation, overall reactant used

Step 8. The carbon radical attacks the hydroxyl group to form the alcohol product and reduce Fe(IV) to Fe(III).

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand

Chemical Components

overall product formed, radical termination, ingold: aromatic bimolecular nucleophilic addition
Download: Image, Marvin File

Step 1. After the substrate binds a water molecule is released from the active site which increases the redox potential. This facilitates the first one electron reduction of Fe(III) to Fe(II) by NADPH (illustrated here as transfer from a general donor).

Step 2. Molecular oxygen binds to Fe(II) to form the oxyferrous intermediate.

Step 3. The second reduction forms a peroxo intermediate.

Step 4. The peroxy oxygen accepts a proton from the C5 hydroxyl group of 6-DEB.

Step 5. A second proton transfer is facilitated by a hydrogen bond network involving Ala241, Ser246 and Glu360.

Step 6. There is fission of the O-O bond and loss of a water molecule. The proton abstraction from 5-OH by O2 transiently forms a substrate alkoxide. After O–O bond fission, the C5 alkoxide is reprotonated, most likely by the solvent.

Step 7. The Fe(IV)-oxo complex undergoes spin inversion to form a radical oxo group which removes a hydrogen from the substrate to form a radical carbon centre and an alcohol group on Fe(IV).

Step 8. The carbon radical attacks the hydroxyl group to form the alcohol product and reduce Fe(IV) to Fe(III).

Products.

Introduction

The catalytic cycle of all cytochrome P450s is conserved. Electrons are delivered to the haem centre by NADH, permitting oxygen reduction. Binding of the substrate promotes electron transfer to P450 haem, reducing the iron from Fe(III) to Fe(II). Dioxygen binds and thus Fe(II) is oxidised back to Fe(III). A second electron is passed to the haem forming a superoxide species, followed by donation of two protons. Catalytic water involved in a Glu244 proton transfer pathway, and uniquely the C5 hydroxyl group of 6-DEB, provide the protons to the distil oxygen. This results in the loss of water and the formation of the low spin Fe(IV) oxo complex. Reprotonation of the catalytic water and 6-DEB after protonation of dioxygen occurs via a hydrogen bonded network presumed to be in contact with the bulk solvent during the course of the reaction. The rebound mechanism forms the alcohol group on the substrate once the oxo group is formed. The Fe(IV)-oxo complex undergoes spin inversion to form a radical oxo group which removes a hydrogen from the substrate to form a radical carbon centre and an alcohol group on Fe(IV). The new radical then attacks the oxygen to form the alcohol group and Fe(III), completing the catalytic cycle.

Catalytic Residues Roles

UniProt PDB* (1oxa)
Glu244 Glu244(243)A Glu244 is involved in the first proton transfer that converts the peroxo to the hydroperoxo intermediate. hydrogen bond donor, proton acceptor, proton donor, proton relay, electrostatic stabiliser
Cys351 Cys351(350)A Coordinated to the Fe ion in heme b. covalently attached, 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

redox reaction, electron transfer, decoordination from a metal ion, coordination to a metal ion, overall reactant used, proton transfer, proton relay, homolysis, overall product formed, radical formation, radical termination, native state of enzyme regenerated

References

  1. Sen K et al. (2014), J Phys Chem B, 118, 2810-2820. Role of Two Alternate Water Networks in Compound I Formation in P450eryF. DOI:10.1021/jp411272h. PMID:24564366.
  2. Nagano S et al. (2005), J Biol Chem, 280, 22102-22107. Crystal Structures of the Ferrous Dioxygen Complex of Wild-type Cytochrome P450eryF and Its Mutants, A245S and A245T: INVESTIGATION OF THE PROTON TRANSFER SYSTEM IN P450eryF. DOI:10.1074/jbc.m501732200. PMID:15824115.
  3. Cupp-Vickery JR et al. (1996), Nat Struct Biol, 3, 632-637. Substrate-assisted catalysis in cytochrome P450eryF. DOI:10.1038/nsb0796-632. PMID:8673608.

Step 1. After the substrate binds a water molecule is released from the active site which increases the redox potential. This facilitates the first one electron reduction of Fe(III) to Fe(II) by NADPH. Electron transfer is illustrated from a general donor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand
Glu244(243)A electrostatic stabiliser, hydrogen bond donor

Chemical Components

redox reaction, electron transfer, decoordination from a metal ion

Step 2. Molecular oxygen binds to Fe(II) to form the oxyferrous intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand
Glu244(243)A hydrogen bond donor, electrostatic stabiliser

Chemical Components

redox reaction, coordination to a metal ion, overall reactant used

Step 3. The second reduction forms a peroxo intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand

Chemical Components

redox reaction, electron transfer

Step 4. The peroxy oxygen accepts a proton from the C5 hydroxyl group of 6-DEB.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand
Glu244(243)A hydrogen bond donor, electrostatic stabiliser

Chemical Components

proton transfer

Step 5. A second proton transfer from Glu244 occurs. Glu244 is well connected to bulk water and can easily replenish its proton from bulk water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand
Glu244(243)A proton relay, proton acceptor, proton donor

Chemical Components

proton transfer, proton relay

Step 6. The O-O bond breaks, releasing water from the metal centre and Fe(III) is oxidised to Fe(IV). The C5 hydroxyl group of DEB is reprotonated. This is likely to be by the solvent.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand
Glu244(243)A electrostatic stabiliser

Chemical Components

homolysis, overall product formed, proton transfer, electron transfer, redox reaction

Step 7. The Fe(IV)-oxo complex undergoes spin inversion to form a radical oxo group which removes a hydrogen from the substrate to form a radical carbon centre and an alcohol group on Fe(IV).

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A metal ligand

Chemical Components

radical formation

Step 8. The carbon radical attacks the hydroxyl group to form the alcohol product and reduce Fe(IV) to Fe(III).

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys351(350)A covalently attached

Chemical Components

radical termination, overall product formed, native state of enzyme regenerated, redox reaction
Download: Image, Marvin File

Step 1. After the substrate binds a water molecule is released from the active site which increases the redox potential. This facilitates the first one electron reduction of Fe(III) to Fe(II) by NADPH. Electron transfer is illustrated from a general donor.

Step 2. Molecular oxygen binds to Fe(II) to form the oxyferrous intermediate.

Step 3. The second reduction forms a peroxo intermediate.

Step 4. The peroxy oxygen accepts a proton from the C5 hydroxyl group of 6-DEB.

Step 5. A second proton transfer from Glu244 occurs. Glu244 is well connected to bulk water and can easily replenish its proton from bulk water.

Step 6. The O-O bond breaks, releasing water from the metal centre and Fe(III) is oxidised to Fe(IV). The C5 hydroxyl group of DEB is reprotonated. This is likely to be by the solvent.

Step 7. The Fe(IV)-oxo complex undergoes spin inversion to form a radical oxo group which removes a hydrogen from the substrate to form a radical carbon centre and an alcohol group on Fe(IV).

Step 8. The carbon radical attacks the hydroxyl group to form the alcohol product and reduce Fe(IV) to Fe(III).

Products.

Contributors

Gemma L. Holliday, Amelia Brasnett