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Information on EC 1.7.1.13 - preQ1 synthase
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1.7.1.13 preQ1 synthaseIUBMB Comments
The reaction occurs in the reverse direction. This enzyme catalyses one of the early steps in the synthesis of queuosine (Q-tRNA), and is followed by the action of EC 2.4.2.29, tRNA-guanosine34 transglycosylase. Queuosine is found in the wobble position of tRNAGUN in Eukarya and Bacteria and is thought to be involved in translational modulation. The enzyme is not a GTP cyclohydrolase, as was thought previously based on sequence-homology studies.
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The enzyme appears in viruses and cellular organisms
Synonyms
nitrile reductase, 7-cyano-7-deazaguanine reductase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
7-cyano-7-deazaguanine reductase
NADPH-dependent 7-cyano-7-deazaguanine reductase
nitrile reductase
NRed
QueF
queuine synthase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
7-aminomethyl-7-carbaguanine + 2 NADP+ = 7-cyano-7-carbaguanine + 2 NADPH + 2 H+
This enzyme catalyses one of the early steps in the synthesis of queosine (Q-tRNA), and is followed by the action of EC 2.4.2.29, queuine tRNA-ribosyltransferase. Queosine is found in the wobble position of tRNAGUN in Eukarya and Bacteria [2] and is thought to be involved in translational modulation. The enzyme is not a GTP cyclohydrolase, as was thought previously based on sequence-homology studies.
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SYSTEMATIC NAME
IUBMB Comments
7-aminomethyl-7-carbaguanine:NADP+ oxidoreductase
The reaction occurs in the reverse direction. This enzyme catalyses one of the early steps in the synthesis of queuosine (Q-tRNA), and is followed by the action of EC 2.4.2.29, tRNA-guanosine34 transglycosylase. Queuosine is found in the wobble position of tRNAGUN in Eukarya and Bacteria [2] and is thought to be involved in translational modulation. The enzyme is not a GTP cyclohydrolase, as was thought previously based on sequence-homology studies.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one + 2 NADPH + 2 H+
2-amino-5-amino-methyl-pyrrolo[2,3-d]pyrimidin-4-one + 2 NADP+
natural substrate
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-
?
2-amino-5-cyanopyrrolo[2,3-d]pyrimidine + 2 NADPH + 2 H+
2-amino-5-amino-methyl-pyrrolo[2,3-d]pyrimidine + 2 NADP+
analogue of natural substrate, poor substrate
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-
?
5-cyanopyrrolo[2,3-d]pyrimidin-4-one + 2 NADPH + 2 H+
5-amino-methyl-pyrrolo[2,3-d]pyrimidin-4-one + 2 NADP+
analogue of natural substrate
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-
?
7-cyano-7-carba-2-deaminoguanine + 2 NADPH + 2 H+
7-aminomethyl-7-carba-2-deaminoguanine + 2 NADP+
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-
-
-
?
7-cyano-7-deazaguanine + 2 NADPH + 2 H+
7-aminomethyl-7-deazaguanine + 2 NADP+
i.e. preQ0
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-
?
7-cyano-7-carbaguanine + 2 NADPH + 2 H+
7-aminomethyl-7-carbaguanine + 2 NADP+
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-
-
-
?
7-cyano-7-carbaguanine + 2 NADPH + 2 H+
7-aminomethyl-7-carbaguanine + 2 NADP+
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-
-
?
7-cyano-7-carbaguanine + 2 NADPH + 2 H+
7-aminomethyl-7-carbaguanine + 2 NADP+
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-
-
?
7-cyano-7-carbaguanine + 2 NADPH + 2 H+
7-aminomethyl-7-carbaguanine + 2 NADP+
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-
?
7-cyano-7-carbaguanine + 2 NADPH + 2 H+
7-aminomethyl-7-carbaguanine + 2 NADP+
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?
7-cyano-7-carbaguanine + 2 NADPH + 2 H+
7-aminomethyl-7-carbaguanine + 2 NADP+
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-
-
?
7-cyano-7-deazaguanine + 2 NADPH + 2 H+
queuine + 2 NADP+
-
late step in biosynthesis of the modified tRNA nucleoside queuosine
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-
?
7-cyano-7-deazaguanine + 2 NADPH + 2 H+
queuine + 2 NADP+
late step in biosynthesis of the modified tRNA nucleoside queuosine
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-
?
7-cyano-7-deazaguanine + 2 NADPH + 2 H+
queuine + 2 NADP+
-
late step in biosynthesis of the modified tRNA nucleoside queuosine
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-
?
7-cyano-7-deazaguanine + 2 NADPH + 2 H+
queuine + 2 NADP+
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queuine is 7-aminomethyl-7-deazaguanine
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?
7-cyano-7-deazaguanine + 2 NADPH + 2 H+
queuine + 2 NADP+
-
queuine is 7-aminomethyl-7-deazaguanine
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?
additional information
?
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no substrates: acetonitrile, benzonitrile and benzylcyanide
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?
additional information
?
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during catalysis each active site of the dimeric enzyme binds one substrate molecule. NADPH binds independent of the substrate. The PreQ0 binding pocket of the active site is not involved in the binding of NADPH
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?
additional information
?
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QueF binds substrate preQ0 in a strongly exothermic process (DeltaH 80.3 kJ/mol) whereby the thioimide adduct is formed with half-of-the-sites reactivity in the homodimeric enzyme. Both steps of preQ0 reduction involve transfer of the 4-pro-R-hydrogen from NADPH. They proceed about 4-7fold more slowly than trapping of the enzyme-bound preQ0 as covalent thioimide and are mainly rate-limiting for the enzyme's kcat
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?
additional information
?
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enzyme is highly specific for substrate preQ0
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?
additional information
?
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enzyme is highly specific for substrate preQ0
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?
additional information
?
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the nitrile to amine conversion proceeds through four major stages: formation of a C-S covalent bond between the substrate and the catalytic cysteine residue to form the thioimidate intermediate, hydride transfer from NADPH to the substrate to generate the thiohemiaminal intermediate, cleavage of the C-S covalent bond to generate the imine intermediate, and second hydride transfer from NADPH to the imine intermediate to generate the final amine product. The free energy barrier for the rate-limiting step, i.e. the second hydride transfer, is20.8 kcal/mol
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?
additional information
?
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the nitrile to amine conversion proceeds through four major stages: formation of a C-S covalent bond between the substrate and the catalytic cysteine residue to form the thioimidate intermediate, hydride transfer from NADPH to the substrate to generate the thiohemiaminal intermediate, cleavage of the C-S covalent bond to generate the imine intermediate, and second hydride transfer from NADPH to the imine intermediate to generate the final amine product. The free energy barrier for the rate-limiting step, i.e. the second hydride transfer, is20.8 kcal/mol
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?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one + 2 NADPH + 2 H+
2-amino-5-amino-methyl-pyrrolo[2,3-d]pyrimidin-4-one + 2 NADP+
natural substrate
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-
?
7-cyano-7-deazaguanine + 2 NADPH + 2 H+
7-aminomethyl-7-deazaguanine + 2 NADP+
i.e. preQ0
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-
?
7-cyano-7-deazaguanine + 2 NADPH + 2 H+
queuine + 2 NADP+
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late step in biosynthesis of the modified tRNA nucleoside queuosine
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-
?
7-cyano-7-deazaguanine + 2 NADPH + 2 H+
queuine + 2 NADP+
late step in biosynthesis of the modified tRNA nucleoside queuosine
-
-
?
7-cyano-7-deazaguanine + 2 NADPH + 2 H+
queuine + 2 NADP+
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late step in biosynthesis of the modified tRNA nucleoside queuosine
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-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
7-formyl-7-deazaguanine
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carbonyl analogue of the imine intermediate, recognized by QueF as weak ligand for binding but not as substrate for reduction or oxidation
peroxide
peroxide-induced inactivation of the wild-type enzyme is reversible with thioredoxin
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0061
2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one
wild-type, pH 7.5, 25°C
0.00025
7-cyano-7-carbaguanine
below 0.25 microM, pH 7.4, 30°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.11
2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one
wild-type, pH 7.5, 25°C
0.024
7-cyano-7-carbaguanine
pH 7.4, 30°C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
10.8
substrate 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one, mutant F228W, pH 7.5, 25°C
117
substrate 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one, wild-type, pH 7.5, 25°C
12.9
substrate 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one, mutant S90A, pH 7.5, 25°C
14.3
substrate 5-cyanopyrrolo[2,3-d]pyrimidin-4-one, mutant H229A, pH 7.5, 25°C
17.9
substrate 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one, mutant H229A, pH 7.5, 25°C
3
substrate 5-cyanopyrrolo[2,3-d]pyrimidin-4-one, mutant E230Q, pH 7.5, 25°C
3.8
substrate 5-cyanopyrrolo[2,3-d]pyrimidin-4-one, mutant S90A, pH 7.5, 25°C
4.4
substrate 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one, mutant E230Q, pH 7.5, 25°C
8.7
substrate 5-cyanopyrrolo[2,3-d]pyrimidin-4-one, mutant F228W, pH 7.5, 25°C
90
substrate 5-cyanopyrrolo[2,3-d]pyrimidin-4-one, wild-type, pH 7.5, 25°C
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.5 - 8
more than 60% of maximum activity
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Show AA Sequence and Transmembrane Helices (21201 entries)
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homodimer
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
sitting-drop vapor-diffusion method, space group: P3(1)21. Unit-cell parameters: a = b = 93.52 A, c = 193.76 A
structure of mutant E97Q, displays an intramolecular disulfide formed between the catalytic Cys55 and a conserved Cys99 located near the active site. This structure exhibits major rearrangement of the loops responsible for substrate binding
construction of homology model and docking studies of natural and non-natural substrates. Residues C190 and D197 play an essential role in the catalytic mechanism
homology model based on the crystal structure of nitrile reductase from Vibrio cholerae, PDB ID 3uxv. The side chain of residue Glu89 and the main chain of Ser90 form hydrogen bonds with the ring nitrogen atoms of the substrate. The carboxylate group of Glu230 also forms hydrogen bonds with the substrate preQ0. The aromatic ring of Phe228 may form a pi-pi-stacking interaction with the aromatic ring of preQ0, while the main chain of His229coordinates the keto-functionality of the substrate
hanging drop vapor diffusion method, using 0.04 M sodium dihydrogen phosphate, 0.96 M dipotassium hydrogen phosphate, and 10 mM GTP
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C99A
mutant does not compromise enzyme activity. Contrary to wild-type, peroxide-induced inactivation is irreversible
C99S
mutant does not compromise enzyme activity. Contrary to wild-type, peroxide-induced inactivation is irreversible
C99A
-
mutant does not compromise enzyme activity. Contrary to wild-type, peroxide-induced inactivation is irreversible
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C99S
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mutant does not compromise enzyme activity. Contrary to wild-type, peroxide-induced inactivation is irreversible
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C190A
C190S
the mutation annihilates preQ0 covalent binding and largely disrupts the nitrile-to-amine reductase activity
D197A
the variant does not take up protons in conjunction with preQ0 binding
D197H
the variant recovers proton uptake to a level almost analogous to that seen with the wildtype enzyme
E230Q
about 4% of wild-type activity for natural substrate 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one
E89A
the variant performs primarily (more than 90%) a two-electron reduction of 7-cyano-7-deazaguanine (preQ0), releasing hydrolyzed imine (7-formyl-7-deazaguanine) as the product
E89L
the variant performs primarily (more than 90%) a two-electron reduction of 7-cyano-7-deazaguanine (preQ0), releasing hydrolyzed imine (7-formyl-7-deazaguanine) as the product
F228A
the variant performs primarily (more than 90%) a two-electron reduction of 7-cyano-7-deazaguanine (preQ0), releasing hydrolyzed imine (7-formyl-7-deazaguanine) as the product
F228W
about 8% of wild-type activity for natural substrate 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one
H229A
I192A
the 7-cyano-7-deazaguanine (preQ0) reduction by the mutant gives 7-aminomethyl-7-deazaguanine (preQ1) and 7-formyl-7-deazaguanine at a 1:1 ratio
L191A
the 7-cyano-7-deazaguanine (preQ0) reduction by the mutant gives 7-aminomethyl-7-deazaguanine (preQ1) and 7-formyl-7-deazaguanine at a 4:1 ratio
S90A
about 10% of wild-type activity for natural substrate 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one
C190A
-
mutation in catalytic residue, no evidence of covalent binding of substrate preQ0. Mutant displays proton uptake
C190A
the mutation annihilates preQ0 covalent binding and largely disrupts the nitrile-to-amine reductase activity
H229A
about 15% of wild-type activity for natural substrate 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one
H229A
the variant readily forms the thioimidate adduct and is 24fold less active for preQ0 reduction than wild type enzyme
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
immobilized metal ion affinity column chromatography and gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
overproduction in Escherichia coli
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Swairjo, M.A.; Reddy, R.R.; Lee, B.; Van Lanen, S.G.; Brown, S.; de Crecy-Lagard, V.; Iwata-Reuyl, D.; Schimmel, P.
Crystallization and preliminary x-ray characterization of the nitrile reductase QueF: a queuosine-biosynthesis enzyme
Acta Crystallogr. Sect. F
F61
945-948
2005
Bacillus subtilis (O31678), Bacillus subtilis
brenda
Kuchino, Y.; Kasai, H.; Nihei, K.; Nishimura, S.
Biosynthesis of the modified nucleoside Q in transfer RNA
Nucleic Acids Res.
3
393-398
1976
Salmonella enterica subsp. enterica serovar Typhimurium
brenda
Okada, N.; Noguchi, S.; Nishimura, S.; Ohgi, T.; Goto, T.; Crain, P.F.; McCloskey, J.A.
Structure determination of a nucleoside Q precursor isolated from E. coli tRNA: 7-(aminomethyl)-7-deazaguanosine
Nucleic Acids Res.
5
2289-2296
1978
Escherichia coli
brenda
Noguchi, S.; Yamaizumi, Z.; Ohgi, T.; Goto, T.; Nishimura, Y.; Hirota, Y.; Nishimura, S.
Isolation of Q nucleoside precursor present in tRNA of an E. coli mutant and its characterization as 7-(cyano)-7-deazaguanosine
Nucleic Acids Res.
5
4215-4223
1978
Escherichia coli
brenda
van Lanen, S.G.; Reader, J.S.; Swairjo, M.A.; de Crecy-Lagard, V.; Lee, B.; Iwata-Reuyl, D.
From cyclohydrolase to oxidoreductase: discovery of nitrile reductase activity in a common fold
Proc. Natl. Acad. Sci. USA
102
4264-4269
2005
Bacillus subtilis, Escherichia coli
brenda
Kim, Y.; Zhou, M.; Moy, S.; Morales, J.; Cunningham, M.A.; Joachimiak, A.
High-resolution structure of the nitrile reductase QueF combined with molecular simulations provide insight into enzyme mechanism
J. Mol. Biol.
404
127-137
2010
Vibrio cholerae (Q9KTK0), Vibrio cholerae, Vibrio cholerae ATCC 39315 (Q9KTK0)
brenda
Wilding, B.; Winkler, M.; Petschacher, B.; Kratzer, R.; Egger, S.; Steinkellner, G.; Lyskowski, A.; Nidetzky, B.; Gruber, K.; Klempier, N.
Targeting the substrate binding site of E. coli nitrile reductase QueF by modeling, substrate and enzyme engineering
Chemistry
19
7007-7012
2013
Escherichia coli (Q46920), Escherichia coli
brenda
Moeller, K.; Nguyen, G.S.; Hollmann, F.; Hanefeld, U.
Expression and characterization of the nitrile reductase queF from E. coli
Enzyme Microb. Technol.
52
129-133
2013
Escherichia coli (Q46920)
brenda
Ribeiro, A.; Yang, L.; Ramos, M.; Fernandes, P.; Liang, Z.; Hirao, H.
Insight into enzymatic nitrile reduction QM/MM study of the catalytic mechanism of QueF nitrile reductase
ACS Catal.
5
3740-3751
2015
Vibrio cholerae O1 (Q9KTK0), Vibrio cholerae O1 ATCC 39315 (Q9KTK0)
-
brenda
Mohammad, A.; Bon Ramos, A.; Lee, B.W.; Cohen, S.W.; Kiani, M.K.; Iwata-Reuyl, D.; Stec, B.; Swairjo, M.A.
Protection of the queuosine biosynthesis enzyme QueF from irreversible oxidation by a conserved intramolecular disulfide
Biomolecules
7
E30
2017
Bacillus subtilis (O31678), Bacillus subtilis 168 (O31678)
brenda
Gjonaj, L.; Pinkse, M.; Fernandez-Fueyo, E.; Hollmann, F.; Hanefeld, U.
Substrate and cofactor binding to nitrile reductase A mass spectrometry based study
Catal. Sci. Technol.
6
7391-7397
2016
Escherichia coli (Q46920)
-
brenda
Jung, J.; Czabany, T.; Wilding, B.; Klempier, N.; Nidetzky, B.
Kinetic analysis and probing with substrate analogues of the reaction pathway of the nitrile reductase QueF from Escherichia coli