Cloned (Comment) | Organism |
---|---|
recombinant expression of His8-MBP-tagged wild-type and mutant K184A enzymes, as well as seleno-L-methionine-labeled enzyme in Escherichia coli TOP10 cells | Nocardia farcinica |
Crystallization (Comment) | Organism |
---|---|
purified recombinant detagged wild-type and seleno-L-methionine-labeled enzyme mutant K184A, method screening, drop vapor diffusion method, mixing of 0.001 ml of 6 mg/ml protein in 25 mM HEPES/NaOH, pH 7.5, 500 mM NaCl, and 5 mM NADP+ in the presence of either L-Lys or D-Lys, with 0.001 ml of reservoir solution containing 10% w/v PEG 6000, 5% v/v 2,4-methylpentanediol, and 0.1 M HEPES/NaOH, pH 7.5, 3-4 days at 20°C, micro-seeding technique, X-ray diffraction structure determination and analysis at 2.4-2.9 A resolution | Nocardia farcinica |
Protein Variants | Comment | Organism |
---|---|---|
K184A | site-directed mutagenesis | Nocardia farcinica |
K200A | site-directed mutagenesis | Nocardia farcinica |
K202A | site-directed mutagenesis | Nocardia farcinica |
K64A | site-directed mutagenesis, the K64A variant support a conserved amino acid substrate binding site among members of the NMO group of enzymes, crystal structure analysis, overview | Nocardia farcinica |
P238R | site-directed mutagenesis, substitution of Pro to an Arg at position 238 converts NbtG into a NADPH-specific monooxygenase but increases the Km value for NADPH. The P238R enzyme is as uncoupled as wild-type NbtG | Nocardia farcinica |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten steady-state kinetics, determined by oxygen consumption assay or L-lysine hydroxylation assay | Nocardia farcinica | |
0.14 | - |
NADPH | pH 7.5, 25°C, recombinant wild-type enzyme, with L-Lys | Nocardia farcinica | |
0.4 | - |
NADH | pH 7.5, 25°C, recombinant wild-type enzyme, with L-Lys | Nocardia farcinica | |
0.51 | - |
NADPH | pH 7.5, 25°C, recombinant wild-type enzyme, with D-Lys | Nocardia farcinica | |
0.7 | - |
NADPH | pH 7.5, 25°C, recombinant mutant P238R, L-lysine hydroxylation | Nocardia farcinica | |
0.8 | - |
NADPH | pH 7.5, 25°C, recombinant mutant P238R, O2 consumption | Nocardia farcinica | |
1.6 | - |
NADH | pH 7.5, 25°C, recombinant wild-type enzyme, with D-Lys | Nocardia farcinica | |
13 | - |
L-lysine | pH 7.5, 25°C, recombinant mutant K64A, with NADPH | Nocardia farcinica | |
40 | - |
L-lysine | pH 7.5, 25°C, recombinant mutant K64A, with NADH | Nocardia farcinica | |
49 | - |
D-Lysine | pH 7.5, 25°C, recombinant mutant K64A, with NADPH | Nocardia farcinica | |
62 | - |
D-Lysine | pH 7.5, 25°C, recombinant mutant K64A, with NADH | Nocardia farcinica |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-lysine + NADH + H+ + O2 | Nocardia farcinica | - |
N6-hydroxy-L-lysine + NAD+ + H2O | - |
? | |
L-lysine + NADPH + H+ + O2 | Nocardia farcinica | - |
N6-hydroxy-L-lysine + NADP+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Nocardia farcinica | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His8-MBP-tagged wild-type and mutant K184A enzymes, as well as seleno-L-methionine-labeled enzyme from Escherichia coli TOP10 cells by nickel affinity chromatography, dialysis, tag cleavage by TEV protease, followed by a second step of nickel affinity chromatography and by MBP-tracking affinity chromatography, eluting the detagged enzyme, followed by dialysis | Nocardia farcinica |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
L-lysine + NADPH + H+ + O2 = N6-hydroxy-L-lysine + NADP+ + H2O | via C4a-hydroperoxyflavin intermediate | Nocardia farcinica |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-lysine + NADH + H+ + O2 | - |
Nocardia farcinica | N6-hydroxy-D-lysine + NAD+ + H2O | - |
? | |
D-lysine + NADPH + H+ + O2 | - |
Nocardia farcinica | N6-hydroxy-D-lysine + NADP+ + H2O | - |
? | |
L-lysine + NADH + H+ + O2 | - |
Nocardia farcinica | N6-hydroxy-L-lysine + NAD+ + H2O | - |
? | |
L-lysine + NADPH + H+ + O2 | - |
Nocardia farcinica | N6-hydroxy-L-lysine + NADP+ + H2O | - |
? | |
additional information | NbtG is unable to stabilize the FADOOH intermediate, which results in production of hydrogen peroxide and superoxide. NbtG is also active on D-Lys, although it binds L-Lys with a higher affinity. NbtG can use both NADH and NADPH and is highly uncoupled, producing more superoxide and hydrogen peroxide than hydroxylated Lys. NbtG is highly active in the absence of L-Lys, having about 2fold higher activity with NADPH as compared with NADH. Under these conditions, NbtG functions as an oxidase, as no hydroxylation occurs, overview | Nocardia farcinica | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
flavin-dependent lysine monooxygenase | - |
Nocardia farcinica |
lysine monooxygenase | - |
Nocardia farcinica |
N-hydroxylating monooxygenase | - |
Nocardia farcinica |
NbtG | - |
Nocardia farcinica |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Nocardia farcinica |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.021 | - |
D-Lysine | pH 7.5, 25°C, recombinant mutant K64A, with NADH | Nocardia farcinica | |
0.13 | - |
D-Lysine | pH 7.5, 25°C, recombinant mutant K64A, with NADPH | Nocardia farcinica | |
0.146 | - |
L-lysine | pH 7.5, 25°C, recombinant mutant P238R, with NADPH | Nocardia farcinica | |
0.18 | - |
L-lysine | pH 7.5, 25°C, recombinant mutant K64A, with NADH | Nocardia farcinica | |
0.34 | - |
L-lysine | pH 7.5, 25°C, recombinant mutant K64A, with NADPH | Nocardia farcinica | |
0.6 | - |
O2 | pH 7.5, 25°C, recombinant mutant P238R, with NADPH | Nocardia farcinica | |
1.08 | - |
NADPH | pH 7.5, 25°C, recombinant wild-type enzyme, with L-Lys | Nocardia farcinica | |
1.29 | - |
NADH | pH 7.5, 25°C, recombinant wild-type enzyme, with L-Lys | Nocardia farcinica | |
2.5 | - |
NADH | pH 7.5, 25°C, recombinant wild-type enzyme, with D-Lys | Nocardia farcinica | |
2.9 | - |
NADPH | pH 7.5, 25°C, recombinant wild-type enzyme, with D-Lys | Nocardia farcinica |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Nocardia farcinica |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Nocardia farcinica | |
additional information | an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the flavin adenine dinucleotide (FAD) domain precludes binding of the nicotinamide cofactor | Nocardia farcinica | |
NADH | - |
Nocardia farcinica | |
NADPH | - |
Nocardia farcinica |
General Information | Comment | Organism |
---|---|---|
additional information | an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the flavin adenine dinucleotide (FAD) domain precludes binding of the nicotinamide cofactor | Nocardia farcinica |
physiological function | the flavoenzyme catalyzes the NADPH-and oxygen-dependent hydroxylation of lysine, NbtG utilizes NADPH and molecular oxygen to hydroxylate the Nepsilon-atom of L-Lys. NbtG is unable to stabilize the FADOOH intermediate, which results in production of hydrogen peroxide and superoxide. The biosynthesis of the siderophore nocobactin in Nocardia farcinica requires functioning of NbtG | Nocardia farcinica |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00034 | - |
D-Lysine | pH 7.5, 25°C, recombinant mutant K64A, with NADH | Nocardia farcinica | |
0.0027 | - |
D-Lysine | pH 7.5, 25°C, recombinant mutant K64A, with NADPH | Nocardia farcinica | |
0.0045 | - |
L-lysine | pH 7.5, 25°C, recombinant mutant K64A, with NADH | Nocardia farcinica | |
0.026 | - |
L-lysine | pH 7.5, 25°C, recombinant mutant K64A, with NADPH | Nocardia farcinica | |
0.21 | - |
NADPH | pH 7.5, 25°C, recombinant mutant P238R, L-lysine hydroxylation | Nocardia farcinica | |
0.79 | - |
NADPH | pH 7.5, 25°C, recombinant mutant P238R, O2 consumption | Nocardia farcinica | |
1.563 | - |
D-Lysine | pH 7.5, 25°C, recombinant wild-type enzyme, with NADH | Nocardia farcinica | |
3.225 | - |
L-lysine | pH 7.5, 25°C, recombinant wild-type enzyme, with NADH | Nocardia farcinica | |
5.686 | - |
D-Lysine | pH 7.5, 25°C, recombinant wild-type enzyme, with NADPH | Nocardia farcinica | |
7.714 | - |
L-lysine | pH 7.5, 25°C, recombinant wild-type enzyme, with NADPH | Nocardia farcinica |