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 | 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 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Bordetella bronchiseptica | A0A0H3LKL4 | - |
- |
Bordetella bronchiseptica ATCC BAA-588 | A0A0H3LKL4 | - |
- |
Bordetella bronchiseptica NCTC 13252 | A0A0H3LKL4 | - |
- |
Bordetella bronchiseptica RB50 | A0A0H3LKL4 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Bordetella bronchiseptica |
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 | 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 | Comment | Organism |
---|---|---|
BB1770 | - |
Bordetella bronchiseptica |
NicC | - |
Bordetella bronchiseptica |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Bordetella bronchiseptica |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Bordetella bronchiseptica |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Bordetella bronchiseptica | |
NADH | - |
Bordetella bronchiseptica |
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 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 |