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Literature summary for 1.7.1.6 extracted from

  • Feng, J.; Kweon, O.; Xu, H.; Cerniglia, C.E.; Chen, H.
    Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis (2012), Arch. Biochem. Biophys., 520, 99-107.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.7.1.6

Cloned(Commentary)

Cloned (Comment) Organism
-
 Enterococcus faecalis

Crystallization (Commentary)

Crystallization (Comment) Organism
modeling of structure. Binding mode shows that the benzoic acid moiety of substrate methyl red and the nicotinamide ring of NADH are not parallel to the flavin isoalloxazine ring, but lay against it at angles of about 45 and 35 degrees, respectively. The adenine ribose moiety of NADH is surrounded by loop l2 on chain B and alpha3 on chain A in a typical Rossmann fold Enterococcus faecalis

Protein Variants

Protein Variants Comment Organism
A123F 36% of wild-type activity Enterococcus faecalis
D184G complete loss of activity Enterococcus faecalis
E16G 46% of wild-type activity Enterococcus faecalis
F127G complete loss of activity Enterococcus faecalis
L59G 10% of wild-type activity Enterococcus faecalis
N106A 3% of wild-type activity Enterococcus faecalis
N121A 170% of wild-type activity Enterococcus faecalis
R18G 9% of wild-type activity Enterococcus faecalis
R21G 382% of wild-type activity Enterococcus faecalis
R66A 47% of wild-type activity Enterococcus faecalis
V122Y 14% of wild-type activity Enterococcus faecalis
W62A 35% of wild-type activity Enterococcus faecalis
Y129G complete loss of activity Enterococcus faecalis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.00053
-
 Methyl red mutant R66A, pH 7.0, 22°C Enterococcus faecalis
0.003
-
 NADH mutant W62A, pH 7.0, 22°C Enterococcus faecalis
0.0082
-
 Methyl red mutant N121A, pH 7.0, 22°C Enterococcus faecalis
0.0112
-
 Methyl red mutant N106A, pH 7.0, 22°C Enterococcus faecalis
0.0126
-
 Methyl red mutant R21G, pH 7.0, 22°C Enterococcus faecalis
0.0167
-
 Methyl red mutant R18G, pH 7.0, 22°C Enterococcus faecalis
0.024
-
 Methyl red wild-type, pH 7.0, 22°C Enterococcus faecalis
0.0288
-
 Methyl red mutant L59G, pH 7.0, 22°C Enterococcus faecalis
0.0335
-
 Methyl red mutant W62A, pH 7.0, 22°C Enterococcus faecalis
0.0485
-
 Methyl red mutant A123F, pH 7.0, 22°C Enterococcus faecalis
0.109
-
 Methyl red mutant V122Y, pH 7.0, 22°C Enterococcus faecalis
0.143
-
 NADH wild-type, pH 7.0, 22°C Enterococcus faecalis
0.25
-
 NADH mutant E16G, pH 7.0, 22°C Enterococcus faecalis
0.292
-
 NADH mutant N121A, pH 7.0, 22°C Enterococcus faecalis
0.38
-
 NADH mutant R21G, pH 7.0, 22°C Enterococcus faecalis
0.405
-
 NADH mutant V122Y, pH 7.0, 22°C Enterococcus faecalis
0.411
-
 NADH mutant A123F, pH 7.0, 22°C Enterococcus faecalis
0.581
-
 NADH mutant R66A, pH 7.0, 22°C Enterococcus faecalis
0.624
-
 NADH mutant R18G, pH 7.0, 22°C Enterococcus faecalis
0.793
-
 NADH mutant N106A, pH 7.0, 22°C Enterococcus faecalis
2.298
-
 NADH mutant L59G, pH 7.0, 22°C Enterococcus faecalis

Organism

Organism UniProt Comment Textmining
Enterococcus faecalis Q831B2
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
methyl red + NADH + H+
-
 Enterococcus faecalis ? + NAD+
-
 ?

Synonyms

Synonyms Comment Organism
AzoA
-
 Enterococcus faecalis
AzoR
-
 Enterococcus faecalis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.2
-
 Methyl red mutant N106A, pH 7.0, 22°C Enterococcus faecalis
3.17
-
 Methyl red mutant R18G, pH 7.0, 22°C Enterococcus faecalis
6.61
-
 Methyl red mutant L59G, pH 7.0, 22°C Enterococcus faecalis
17.5
-
 Methyl red mutant V122Y, pH 7.0, 22°C Enterococcus faecalis
20.8
-
 Methyl red mutant E16G, pH 7.0, 22°C Enterococcus faecalis
21.1
-
 Methyl red mutant W62A, pH 7.0, 22°C Enterococcus faecalis
59.1
-
 Methyl red wild-type, pH 7.0, 22°C Enterococcus faecalis
77.3
-
 Methyl red mutant R66A, pH 7.0, 22°C Enterococcus faecalis
111
-
 Methyl red mutant N121A, pH 7.0, 22°C Enterococcus faecalis
140
-
 Methyl red mutant R21G, pH 7.0, 22°C Enterococcus faecalis
245
-
 Methyl red mutant A123F, pH 7.0, 22°C Enterococcus faecalis

Cofactor

Cofactor Comment Organism Structure
FMN
-
 Enterococcus faecalis
NADH
-
 Enterococcus faecalis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.5
-
 NADH mutant N106A, pH 7.0, 22°C Enterococcus faecalis
2.9
-
 NADH mutant L59G, pH 7.0, 22°C Enterococcus faecalis
5
-
 NADH mutant R18G, pH 7.0, 22°C Enterococcus faecalis
44
-
 NADH mutant V122Y, pH 7.0, 22°C Enterococcus faecalis
83
-
 NADH mutant E16G, pH 7.0, 22°C Enterococcus faecalis
110
-
 Methyl red mutant N106A, pH 7.0, 22°C Enterococcus faecalis
111
-
 Methyl red mutant R21G, pH 7.0, 22°C Enterococcus faecalis
130
-
 NADH mutant R66A, pH 7.0, 22°C Enterococcus faecalis
160
-
 Methyl red mutant V122Y, pH 7.0, 22°C Enterococcus faecalis
190
-
 Methyl red mutant R18G, pH 7.0, 22°C Enterococcus faecalis
230
-
 Methyl red mutant L59G, pH 7.0, 22°C Enterococcus faecalis
370
-
 NADH mutant R21G, pH 7.0, 22°C Enterococcus faecalis
380
-
 NADH mutant N121A, pH 7.0, 22°C Enterococcus faecalis
410
-
 NADH wild-type, pH 7.0, 22°C Enterococcus faecalis
600
-
 NADH mutant A123F, pH 7.0, 22°C Enterococcus faecalis
630
-
 Methyl red mutant W62A, pH 7.0, 22°C Enterococcus faecalis
2460
-
 Methyl red wild-type, pH 7.0, 22°C Enterococcus faecalis
5050
-
 Methyl red mutant A123F, pH 7.0, 22°C Enterococcus faecalis
7030
-
 NADH mutant W62A, pH 7.0, 22°C Enterococcus faecalis
13540
-
 Methyl red mutant N121A, pH 7.0, 22°C Enterococcus faecalis
14580
-
 Methyl red mutant R66A, pH 7.0, 22°C Enterococcus faecalis