NADPH-dependent 7-cyano-7-deazaguanine reductase

 

First discovered from the biosynthetic pathway of queuosine, QueF nitrile reductase is part of the tRNA biosynthetic pathway in the cytosol of bacteria. The role of QueF in this pathway is to catalyse the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1) by a unique four-electron reduction of nitrile to amine. This pathway is crucial for the pathogenicity of bacteria and has the potential to be used in industrial catalysis of organic compounds.

 

Reference Protein and Structure

Sequence
Q9KTK0 UniProt (1.7.1.13) IPR016428 (Sequence Homologues) (PDB Homologues)
Biological species
Vibrio cholerae O1 biovar El Tor str. N16961 (Bacteria) Uniprot
PDB
3uxj - Crystal Structure of 7-cyano-7-deazaguanine reductase, QueF from Vibrio cholerae complexed with NADP and PreQ0 (1.401 Å) PDBe PDBsum 3uxj
Catalytic CATH Domains
3.30.1130.10 CATHdb (see all for 3uxj)
Click To Show Structure

Enzyme Reaction (EC:1.7.1.13)

7-cyano-7-deazaguanine
CHEBI:45075ChEBI
+
hydron
CHEBI:15378ChEBI
+
NADPH(4-)
CHEBI:57783ChEBI
→
7-ammoniomethyl-7-deazaguanine
CHEBI:58703ChEBI
+
NADP(3-)
CHEBI:58349ChEBI
Alternative enzyme names: YkvM, QueF, PreQ(0) reductase, PreQ(0) oxidoreductase, 7-cyano-7-deazaguanine reductase, Queuine:NADP(+) oxidoreductase (incorrect as queuine is not the product), Queuine synthase (incorrect as queuine is not the product),

Enzyme Mechanism

Introduction

The nitrile to amine conversion begins with the orientation of the substrate by seven key residues. The first stage of the mechanism is the activation of Cys194 by Asp201 which subsequently attacks the carbon of the nitrile group to form a C-S covalent bond. Secondly, NADPH facilitates a hydride transfer to the thoimidate intermediate. Then, following cleavage of the C-S bond a second hydride transfer occurs by a second NADPH molecule in the active site, forming the amine product.

Catalytic Residues Roles

UniProt PDB* (3uxj)
Cys188 Cys194(197)A Nucleophile in the reaction, facilitating hydride transfer to the thioimidate intermediate. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor
Asp195 Asp201(204)A Activates Cys194 and stabilises charge in the nitrile group along the reaction. activator, proton acceptor, proton donor
His227 His233(236)A Stabilises charge of Asp201. electrostatic stabiliser
Thr191 Thr197(200)A Stabilises negative charge of Cys194 following activation. electrostatic stabiliser
Glu228 Glu234(237)A Stabilises positive charge in the active site after hydride transfer. electrostatic stabiliser
*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 addition, intermediate formation, inferred reaction step, hydride transfer, rate-determining step, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed

References

  1. Ribeiro AJM et al. (2015), ACS Catal, 5, 3740-3751. Insight into Enzymatic Nitrile Reduction: QM/MM Study of the Catalytic Mechanism of QueF Nitrile Reductase. DOI:10.1021/acscatal.5b00528.
  2. Jung J et al. (2018), J Biol Chem, 293, 3720-3733. Evidence of a sequestered imine intermediate during reduction of nitrile to amine by the nitrile reductase QueF from Escherichia coli. DOI:10.1074/jbc.M117.804583. PMID:29339556.
  3. Jung J et al. (2016), J Biol Chem, 291, 25411-25426. Kinetic Analysis and Probing with Substrate Analogues of the Reaction Pathway of the Nitrile Reductase QueF from Escherichia coli. DOI:10.1074/jbc.M116.747014. PMID:27754868.
  4. Wilding B et al. (2013), Chemistry, 19, 7007-7012. Targeting the substrate binding site of E. coli nitrile reductase QueF by modeling, substrate and enzyme engineering. DOI:10.1002/chem.201300163. PMID:23595998.
  5. Kim Y et al. (2010), J Mol Biol, 404, 127-137. High-resolution structure of the nitrile reductase QueF combined with molecular simulations provide insight into enzyme mechanism. DOI:10.1016/j.jmb.2010.09.042. PMID:20875425.
  6. Lee BW et al. (2007), Biochemistry, 46, 12844-12854. Mechanistic studies of Bacillus subtilis QueF, the nitrile oxidoreductase involved in queuosine biosynthesis. DOI:10.1021/bi701265r. PMID:17929836.
  7. Crystal Structure of 7-cyano-7-deazaguanine reductase, QueF from Vibrio cholerae complexed with NADP and PreQ0. DOI:10.2210/pdb3uxj/pdb.

Step 1. Asp201 activates Cys194 by deprotonation.

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Catalytic Residues Roles

Residue Roles
Asp201(204)A proton acceptor
Cys194(197)A proton donor
Thr197(200)A electrostatic stabiliser
Asp201(204)A activator
His233(236)A electrostatic stabiliser

Chemical Components

proton transfer

Step 2. Cys194 performs a nucleophilic attack on the nitrile carbon, forming a thioimidate intermediate.

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Catalytic Residues Roles

Residue Roles
Cys194(197)A nucleophile
Asp201(204)A proton donor
Thr197(200)A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, intermediate formation

Step 3. Protonation of Asp201 and the substrate; inferred by curator.

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Catalytic Residues Roles

Residue Roles
Asp201(204)A proton acceptor

Chemical Components

proton transfer, inferred reaction step

Step 4. Hydride transfer from NADPH to substrate.

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Catalytic Residues Roles

Residue Roles
Cys194(197)A covalently attached
Glu234(237)A electrostatic stabiliser

Chemical Components

hydride transfer, rate-determining step

Step 5. Cleavage of C-S covalent bond.

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Catalytic Residues Roles

Residue Roles
Cys194(197)A nucleofuge
Thr197(200)A electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse

Step 6. Cys194 protonation by Asp201.

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Catalytic Residues Roles

Residue Roles
Asp201(204)A proton donor
Cys194(197)A proton acceptor

Chemical Components

proton transfer

Step 7. Hydride transfer from a second NADPH molecule to carbon atom of imine.

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Catalytic Residues Roles

Residue Roles

Chemical Components

hydride transfer, rate-determining step

Step 8. Protonation of amine group by water molecule from bulk solvent. Inferred by curator; protonation may occur outside active site.

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Catalytic Residues Roles

Residue Roles

Chemical Components

proton transfer, inferred reaction step, overall product formed
Download: Image, Marvin File

Step 1. Asp201 activates Cys194 by deprotonation.

Step 2. Cys194 performs a nucleophilic attack on the nitrile carbon, forming a thioimidate intermediate.

Step 3. Protonation of Asp201 and the substrate; inferred by curator.

Step 4. Hydride transfer from NADPH to substrate.

Step 5. Cleavage of C-S covalent bond.

Step 6. Cys194 protonation by Asp201.

Step 7. Hydride transfer from a second NADPH molecule to carbon atom of imine.

Step 8. Protonation of amine group by water molecule from bulk solvent. Inferred by curator; protonation may occur outside active site.

Products.

Contributors

Noa Marson, Trung Nguyen, Marko Babić, Yordanos Abeje, Antonio Ribeiro