Ribonucleoside-diphosphate reductase (class I)

 

Ribonucleoside-diphosphate reductase (RNR) catalyses the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides using thioredoxin as a co-substrate. This entry represents the class I RNRs. Class I enzymes consist of two homodimeric proteins, R1 (alpha2), coded by the nrdA gene, and R2 (beta2), coded by nrdB. The large alpha chain harbours the catalytic site and binding sites for allosteric effectors. The small beta chain contains an oxygen-linked diferric centre and, in its active form, a stable tyrosyl free radical.

 

Reference Protein and Structure

Sequences
P00452 UniProt (1.17.4.1)
P69924 UniProt (1.17.4.1) IPR013346 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
5cnv - Crystal structure of the dATP inhibited E. coli class Ia ribonucleotide reductase complex bound to GDP and TTP at 3.20 Angstroms resolution (3.2 Å) PDBe PDBsum 5cnv
Catalytic CATH Domains
3.20.70.20 CATHdb 1.10.620.20 CATHdb (see all for 5cnv)
Cofactors
Iron(3+) (2)
Click To Show Structure

Enzyme Reaction (EC:1.17.4.1)

L-cystine residue
CHEBI:50058ChEBI
+
2'-deoxyribonucleoside 5'-diphosphate(3-)
CHEBI:73316ChEBI
+
water
CHEBI:15377ChEBI
→
L-cysteine residue
CHEBI:29950ChEBI
+
nucleoside 5'-diphosphate(3-)
CHEBI:57930ChEBI
Alternative enzyme names: 2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, ADP reductase, CDP reductase, UDP reductase, Nucleoside diphosphate reductase, Ribonucleoside 5'-diphosphate reductase, Ribonucleoside diphosphate reductase, Ribonucleotide diphosphate reductase, Ribonucleotide reductase, RR, NrdB (gene name), NrdF (gene name), NrdJ (gene name),

Enzyme Mechanism

Introduction

The reaction is initiated by binding of the substrate into the active site of the reduced enzyme. In the E. coli enzyme this leads to a transfer of the radical function from Y122 of the R2 protein to C439 of the R1 protein, generating a thiol radical. The radical initiates the reduction of the ribonucleotide by abstracting the 3′-hydrogen atom, thereby generating a substrate radical. Radical formation facilitates the leaving of the protonated OH-group at C-2′. A substrate cation radical is generated that subsequently is reduced by the redox-active cysteine pair C225 and C462. Finally the hydrogen atom stored at C439 is returned to C-3′ with regeneration of the thiol radical at C439. E441 and N437 stabilise the interaction between enzyme and substrate by hydrogen bonding to the oxygens at C-3′ and C-2′, respectively.

Catalytic Residues Roles

UniProt PDB* (5cnv)
Tyr123 Tyr122G Present as a tyrosyl radical in the ground state of the enzyme. It is thought to activate the substrate ribose through the formation of a transient substrate radical.
Cys439 Cys439A Forms a cysteine radical intermediate.
Asn437, Glu441 Asn437A, Glu441A Stabilise the reactive intermediates via hydrogen bonding interactions.
Cys225, Cys462 Cys225A, Cys462A Important for hydrogen atom transfer.
Tyr731, Tyr730, Asp238 Tyr731A, Tyr730A, Asp237G In class I enzymes the thiol radical is formed transiently during each catalytic cycle by radical transfer from the stable tyrosyl radical. A specific pathway leading from Y-122 of R2 to C439 of R1 is probably involved. In it participate the iron center; the R2 residues D237, W48, and Y356 (missing in the crystal structure); and the R1 residues Y731 and Y730.
*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

References

  1. Jordan A et al. (1998), Annu Rev Biochem, 67, 71-98. Ribonucleotide reductases. DOI:10.1146/annurev.biochem.67.1.71. PMID:9759483.
  2. Olshansky L et al. (2016), J Am Chem Soc, 138, 1196-1205. Charge-Transfer Dynamics at the α/β Subunit Interface of a Photochemical Ribonucleotide Reductase. DOI:10.1021/jacs.5b09259. PMID:26710997.
  3. Song DY et al. (2015), Chem Sci, 6, 4519-4524. Direct Interfacial Y731 Oxidation in α2 by a Photoβ2 Subunit of E. coli Class Ia Ribonucleotide Reductase. DOI:10.1039/C5SC01125F. PMID:26504513.
  4. Nick TU et al. (2015), J Am Chem Soc, 137, 289-298. Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli α2 ribonucleotide reductase. DOI:10.1021/ja510513z. PMID:25516424.
  5. Wörsdörfer B et al. (2013), J Am Chem Soc, 135, 8585-8593. Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer. DOI:10.1021/ja401342s. PMID:23676140.
  6. Han WG et al. (2011), Dalton Trans, 40, 11164-11175. Mössbauer properties of the diferric cluster and the differential iron(II)-binding affinity of the iron sites in protein R2 of class Ia Escherichia coli ribonucleotide reductase: a DFT/electrostatics study. DOI:10.1039/c1dt10950b. PMID:21837345.
  7. Han WG et al. (2011), Inorg Chem, 50, 2302-2320. DFT calculations for intermediate and active states of the diiron center with a tryptophan or tyrosine radical in Escherichia coli ribonucleotide reductase. DOI:10.1021/ic1020127. PMID:21322584.
  8. Minnihan EC et al. (2011), J Am Chem Soc, 133, 9430-9440. Kinetics of radical intermediate formation and deoxynucleotide production in 3-aminotyrosine-substituted Escherichia coli ribonucleotide reductases. DOI:10.1021/ja201640n. PMID:21612216.
  9. Persson AL et al. (1997), J Biol Chem, 272, 31533-31541. A new mechanism-based radical intermediate in a mutant R1 protein affecting the catalytically essential Glu441 in Escherichia coli ribonucleotide reductase. PMID:9395490.
  10. Ekberg M et al. (1996), J Biol Chem, 271, 20655-20659. Two conserved tyrosine residues in protein R1 participate in an intermolecular electron transfer in ribonucleotide reductase. PMID:8702814.
  11. Logan DT et al. (1996), Structure, 4, 1053-1064. Crystal structure of reduced protein R2 of ribonucleotide reductase: the structural basis for oxygen activation at a dinuclear iron site. DOI:10.1016/s0969-2126(96)00112-8. PMID:8805591.

Introduction

Non native reaction. This is the catalysis reaction of 2′-deoxy-2′-methylidenecytidine-5′-diphosphate ((CH2dCDP) into a 2′-methyl-3′-ketodeoxyribonucleotide, an inhibitor. This happens via a radical transport chain from R2 subunit to R1 subunit. The R1 subunit receives the radical on Cys439 which proceeds to donate it via hydrogen radical transfer to the substrate. The substrate is in concert deprotonated by Glu441. This causes a rearrangement of the radical and intramolecular bonds leading the substrates C6 to deprotonate Cys225 and return the now C2 radical back to Cys439.

Catalytic Residues Roles

UniProt PDB* (5cnv)
Tyr123 Tyr122G Tyr122 is the residue from the R1 subunit that first receives the radical and transfers it to Cys439. single electron relay, pi-pi interaction
Cys225 Cys225A Cys225 protonates the substrates C6 atom after radical transfer. proton donor
Cys439 Cys439A The main reaction molecule is Cys439. It receives the radical from subunit R2 and gives it to the substrate. After substrate rearrangement, deprotonation of Glu441 and protonation of Cys225 the radical is returned to Cys439. hydrogen radical acceptor, hydrogen radical donor, single electron acceptor
Glu441 Glu441A Glu441 deprotonates the substrates C3-OH in concert with radical transfer to C3 by Cys 439. proton acceptor
Tyr731, Tyr730 Tyr731A, Tyr730A Tyr730 and Tyr731 are atoms that transfer the radical created by the FeIII cluster in the R2 subunit to the R1 subunit. This is possible because they are pi-bonded together. single electron relay, pi-pi interaction
*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

radical propagation, hydrogen transfer

References

  1. Perez MA et al. (2010), J Chem Theory Comput, 6, 2770-2781. Understanding the Mechanism for Ribonucleotide Reductase Inactivation by 2'- Deoxy-2'-methylenecytidine-5'-diphosphate. DOI:10.1021/ct1002175. PMID:26616078.
  2. Nick TU et al. (2015), J Am Chem Soc, 137, 289-298. Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli α2 ribonucleotide reductase. DOI:10.1021/ja510513z. PMID:25516424.
  3. Wörsdörfer B et al. (2013), J Am Chem Soc, 135, 8585-8593. Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer. DOI:10.1021/ja401342s. PMID:23676140.

Step 1. First step is a radical transfer from subunit R2 to subunit R1 via pi-bonded tyrosines. The radical ends up on Cys439.

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Catalytic Residues Roles

Residue Roles
Tyr122G single electron relay
Tyr731A single electron relay
Tyr730A single electron relay
Cys439A single electron acceptor
Tyr122G pi-pi interaction
Tyr731A pi-pi interaction
Tyr730A pi-pi interaction

Chemical Components

radical propagation

Step 2. The Cys439 performs a hydrogen radical transfer giving the radical to the C3 atom. This action causes Glu441 to remove a proton from the C3-OH oxygen.

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Catalytic Residues Roles

Residue Roles
Glu441A proton acceptor
Cys439A hydrogen radical acceptor

Chemical Components

radical propagation

Step 3. The CH2dCDP molecule rearranges to form a double bond on the C3-O bond and transfers the radical to C2. This transfer of radical forces the double bond between C2 and C6 to break and Cys225 to donate a proton to the substrate.

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Catalytic Residues Roles

Residue Roles
Cys225A proton donor

Chemical Components

radical propagation, hydrogen transfer

Step 4. The radical is then returned to Cys439 via another hydrogen radical transfer to C2 of the substrate.

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Catalytic Residues Roles

Residue Roles
Cys439A hydrogen radical donor

Chemical Components

radical propagation
Download: Image, Marvin File

Step 1. First step is a radical transfer from subunit R2 to subunit R1 via pi-bonded tyrosines. The radical ends up on Cys439.

Step 2. The Cys439 performs a hydrogen radical transfer giving the radical to the C3 atom. This action causes Glu441 to remove a proton from the C3-OH oxygen.

Step 3. The CH2dCDP molecule rearranges to form a double bond on the C3-O bond and transfers the radical to C2. This transfer of radical forces the double bond between C2 and C6 to break and Cys225 to donate a proton to the substrate.

Step 4. The radical is then returned to Cys439 via another hydrogen radical transfer to C2 of the substrate.

Products.

Contributors

Craig Porter, Gemma L. Holliday, Marko Babić