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Literature summary for 1.14.13.114 extracted from

  • Nakamoto, K.D.; Perkins, S.W.; Campbell, R.G.; Bauerle, M.R.; Gerwig, T.J.; Gerislioglu, S.; Wesdemiotis, C.; Anderson, M.A.; Hicks, K.A.; Snider, M.J.
    Mechanism of 6-hydroxynicotinate 3-monooxygenase, a flavin-dependent decarboxylative hydroxylase involved in bacterial nicotinic acid degradation (2019), Biochemistry, 58, 1751-1763 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene Bb1770, recombinant overexpression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Bordetella bronchiseptica

Protein Variants

Protein Variants Comment Organism
C202A site-directed mutagenesis Bordetella bronchiseptica
H211A site-directed mutagenesis, the mutant shows moderate uncoupling of their rates of NAD+ and 2,5-DHP product formation, suggesting that the variant has lost some efficiency in hydroxylating the substrate Bordetella bronchiseptica
H302A site-directed mutagenesis, the mutant shows moderate uncoupling of their rates of NAD+ and 2,5-DHP product formation, suggesting that the variant has lost some efficiency in hydroxylating the substrate Bordetella bronchiseptica
H47A site-directed mutagenesis, inactive mutant, unable to bind FAD Bordetella bronchiseptica
H47E site-directed mutagenesis, the mutant can bind FAD, but shows very low activity compared to wild-type. The mutant shows significant consequences in its hydroxylating activity, with NADH oxidization proceeding much more rapidly than 6-HNA is decarboxylated and hydroxylated Bordetella bronchiseptica
H47F site-directed mutagenesis, inactive mutant, unable to bind FAD Bordetella bronchiseptica
Y215F site-directed mutagenesis, the mutant shows significant consequences in its hydroxylating activity, with NADH oxidization proceeding much more rapidly than 6-HNA is decarboxylated and hydroxylated Bordetella bronchiseptica
Y225F site-directed mutagenesis, the mutant shows moderate uncoupling of their rates of NAD+ and 2,5-DHP product formation, suggesting that the variant has lost some efficiency in hydroxylating the substrate Bordetella bronchiseptica

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics, comparative steady-state kinetic analysis with different substrates, 13C kinetic isotope effects, wild-type enzyme and mutants, overview. Comparison of the initial steady-state rates of product (2,5-DHP and NAD+) formation measured by HPLC and equilibrium dissociation constants for the NicC-FAD-6HNA complex Bordetella bronchiseptica
0.0028
-
NADH pH 7.5, 25°C, recombinant mutant Y225F Bordetella bronchiseptica
0.0039
-
5-chloro-6-hydroxynicotinate pH 7.5, 25°C, recombinant wild-type enzyme Bordetella bronchiseptica
0.0063
-
NADH pH 7.5, 25°C, recombinant mutant C202A Bordetella bronchiseptica
0.0081
-
NADH pH 7.5, 25°C, recombinant wild-type enzyme Bordetella bronchiseptica
0.019
-
NADH pH 7.5, 25°C, recombinant mutant H302A Bordetella bronchiseptica
0.02
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant H302A Bordetella bronchiseptica
0.025
-
NADH pH 7.5, 25°C, recombinant mutant H211A Bordetella bronchiseptica
0.051
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant Y225F Bordetella bronchiseptica
0.118
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant wild-type enzyme Bordetella bronchiseptica
0.193
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant H211A Bordetella bronchiseptica
0.2
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant C202A Bordetella bronchiseptica
0.31
-
NADH pH 7.5, 25°C, recombinant mutant Y215F Bordetella bronchiseptica
0.6
-
4-hydroxybenzoate pH 7.5, 25°C, recombinant wild-type enzyme Bordetella bronchiseptica
1.6
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant Y215F Bordetella bronchiseptica

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6-hydroxynicotinate + NADH + H+ + O2 Bordetella bronchiseptica
-
2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
?
6-hydroxynicotinate + NADH + H+ + O2 Bordetella bronchiseptica ATCC BAA-588
-
2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
?
6-hydroxynicotinate + NADH + H+ + O2 Bordetella bronchiseptica RB50
-
2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
?
6-hydroxynicotinate + NADH + H+ + O2 Bordetella bronchiseptica NCTC 13252
-
2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
?

Organism

Organism UniProt Comment Textmining
Bordetella bronchiseptica A0A0H3LKL4
-
-
Bordetella bronchiseptica ATCC BAA-588 A0A0H3LKL4
-
-
Bordetella bronchiseptica NCTC 13252 A0A0H3LKL4
-
-
Bordetella bronchiseptica RB50 A0A0H3LKL4
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Bordetella bronchiseptica

Reaction

Reaction Comment Organism Reaction ID
6-hydroxynicotinate + NADH + H+ + O2 = 2,5-dihydroxypyridine + NAD+ + H2O + CO2 reaction mechanism, overview. Determination of an electrophilic aromatic substitution reaction mechanism in which His47-Tyr215 may serve as the general base to catalyze substrate hydroxylation and refine the structural model for substrate binding by NicC Bordetella bronchiseptica

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-hydroxybenzoate + NADH + H+ + O2 the homocyclic analogue of 6-hydroxynicotinate (6-HNA), 4-hydroxybenzoic acid (4-HBA), is decarboxylated and hydroxylated by NicC with a 420fold lower catalytic efficiency than is 6-HNA Bordetella bronchiseptica hydroquinone + NAD+ + H2O + CO2
-
?
4-hydroxybenzoate + NADH + H+ + O2 the homocyclic analogue of 6-hydroxynicotinate (6-HNA), 4-hydroxybenzoic acid (4-HBA), is decarboxylated and hydroxylated by NicC with a 420fold lower catalytic efficiency than is 6-HNA Bordetella bronchiseptica ATCC BAA-588 hydroquinone + NAD+ + H2O + CO2
-
?
4-hydroxybenzoate + NADH + H+ + O2 the homocyclic analogue of 6-hydroxynicotinate (6-HNA), 4-hydroxybenzoic acid (4-HBA), is decarboxylated and hydroxylated by NicC with a 420fold lower catalytic efficiency than is 6-HNA Bordetella bronchiseptica RB50 hydroquinone + NAD+ + H2O + CO2
-
?
4-hydroxybenzoate + NADH + H+ + O2 the homocyclic analogue of 6-hydroxynicotinate (6-HNA), 4-hydroxybenzoic acid (4-HBA), is decarboxylated and hydroxylated by NicC with a 420fold lower catalytic efficiency than is 6-HNA Bordetella bronchiseptica NCTC 13252 hydroquinone + NAD+ + H2O + CO2
-
?
5-chloro-6-hydroxynicotinate + NADH + H+ + O2 substrate analogue 5-chloro-6-HNA is 10fold more catalytically efficient than 6-HNA Bordetella bronchiseptica 4-chloro-2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
?
5-chloro-6-hydroxynicotinate + NADH + H+ + O2 substrate analogue 5-chloro-6-HNA is 10fold more catalytically efficient than 6-HNA Bordetella bronchiseptica ATCC BAA-588 4-chloro-2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
?
5-chloro-6-hydroxynicotinate + NADH + H+ + O2 substrate analogue 5-chloro-6-HNA is 10fold more catalytically efficient than 6-HNA Bordetella bronchiseptica RB50 4-chloro-2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
?
5-chloro-6-hydroxynicotinate + NADH + H+ + O2 substrate analogue 5-chloro-6-HNA is 10fold more catalytically efficient than 6-HNA Bordetella bronchiseptica NCTC 13252 4-chloro-2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
?
6-hydroxynicotinate + NADH + H+ + O2
-
Bordetella bronchiseptica 2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
?
6-hydroxynicotinate + NADH + H+ + O2
-
Bordetella bronchiseptica ATCC BAA-588 2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
?
6-hydroxynicotinate + NADH + H+ + O2
-
Bordetella bronchiseptica RB50 2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
?
6-hydroxynicotinate + NADH + H+ + O2
-
Bordetella bronchiseptica NCTC 13252 2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
?

Synonyms

Synonyms Comment Organism
BB1770
-
Bordetella bronchiseptica
NicC
-
Bordetella bronchiseptica

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Bordetella bronchiseptica

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.074
-
4-hydroxybenzoate pH 7.5, 25°C, recombinant wild-type enzyme Bordetella bronchiseptica
0.26
-
NADH pH 7.5, 25°C, recombinant mutant C202A Bordetella bronchiseptica
0.26
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant C202A Bordetella bronchiseptica
0.9
-
NADH pH 7.5, 25°C, recombinant mutant H302A Bordetella bronchiseptica
0.9
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant H302A Bordetella bronchiseptica
2.18
-
5-chloro-6-hydroxynicotinate pH 7.5, 25°C, recombinant wild-type enzyme Bordetella bronchiseptica
5
-
NADH pH 7.5, 25°C, recombinant wild-type enzyme Bordetella bronchiseptica
5
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant wild-type enzyme Bordetella bronchiseptica
5.24
-
NADH pH 7.5, 25°C, recombinant mutant Y225F Bordetella bronchiseptica
5.24
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant Y225F Bordetella bronchiseptica
5.9
-
NADH pH 7.5, 25°C, recombinant mutant H211A Bordetella bronchiseptica
5.9
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant H211A Bordetella bronchiseptica
12
-
NADH pH 7.5, 25°C, recombinant mutant Y215F Bordetella bronchiseptica
12
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant Y215F Bordetella bronchiseptica

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Bordetella bronchiseptica

Cofactor

Cofactor Comment Organism Structure
FAD
-
Bordetella bronchiseptica
NADH
-
Bordetella bronchiseptica

General Information

General Information Comment Organism
evolution 6-hydroxynicotinate 3-monooxygenase (NicC) is a group A FAD-dependent monooxygenase Bordetella bronchiseptica
additional information residues Tyr215 and His47 are both critical determinants of 6-hydroxynicotinate (6-HNA) binding and in coupling rates of 2,5-dihydroxypyridine (2,5-DHP) and NAD+ product formation. Two mechanistic proposals for the substrate hydroxylation and decarboxylation reaction catalyzed by NicC using the C(4a)-hydroperoxyflavin intermediate (FADHOOH), and determination of an electrophilic aromatic substitution reaction mechanism in which His47-Tyr215 may serve as the general base to catalyze substrate hydroxylation and refine the structural model for substrate binding by NicC. Residues H302, Y215, and H47 are critical determinants of the hydroxylation steps in catalysis Bordetella bronchiseptica
physiological function 6-hydroxynicotinate 3-monooxygenase (NicC) is a group A FAD-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP) with concomitant oxidation of NADH in nicotinic acid degradation by aerobic bacteria Bordetella bronchiseptica

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.123
-
4-hydroxybenzoate pH 7.5, 25°C, recombinant wild-type enzyme Bordetella bronchiseptica
0.26
-
NADH pH 7.5, 25°C, recombinant mutant C202A Bordetella bronchiseptica
0.9
-
NADH pH 7.5, 25°C, recombinant mutant H302A Bordetella bronchiseptica
1.3
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant C202A Bordetella bronchiseptica
5
-
NADH pH 7.5, 25°C, recombinant wild-type enzyme Bordetella bronchiseptica
5.24
-
NADH pH 7.5, 25°C, recombinant mutant Y225F Bordetella bronchiseptica
5.9
-
NADH pH 7.5, 25°C, recombinant mutant H211A Bordetella bronchiseptica
7.5
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant Y215F Bordetella bronchiseptica
12
-
NADH pH 7.5, 25°C, recombinant mutant Y215F Bordetella bronchiseptica
30.6
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant H211A Bordetella bronchiseptica
42.4
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant wild-type enzyme Bordetella bronchiseptica
45
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant H302A Bordetella bronchiseptica
102.8
-
6-Hydroxynicotinate pH 7.5, 25°C, recombinant mutant Y225F Bordetella bronchiseptica
559
-
5-chloro-6-hydroxynicotinate pH 7.5, 25°C, recombinant wild-type enzyme Bordetella bronchiseptica