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Literature summary extracted from

  • Binda, C.; Robinson, R.M.; Martin Del Campo, J.S.; Keul, N.D.; Rodriguez, P.J.; Robinson, H.H.; Mattevi, A.; Sobrado, P.
    An unprecedented NADPH domain conformation in lysine monooxygenase NbtG provides insights into uncoupling of oxygen consumption from substrate hydroxylation (2015), J. Biol. Chem., 290, 12676-12688 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.59 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 (Commentary)

EC Number Crystallization (Comment) Organism
1.14.13.59 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

EC Number Protein Variants Comment Organism
1.14.13.59 K184A site-directed mutagenesis Nocardia farcinica
1.14.13.59 K200A site-directed mutagenesis Nocardia farcinica
1.14.13.59 K202A site-directed mutagenesis Nocardia farcinica
1.14.13.59 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
1.14.13.59 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.59 additional information
-
 additional information Michaelis-Menten steady-state kinetics, determined by oxygen consumption assay or L-lysine hydroxylation assay Nocardia farcinica
1.14.13.59 0.14
-
 NADPH pH 7.5, 25°C, recombinant wild-type enzyme, with L-Lys Nocardia farcinica
1.14.13.59 0.4
-
 NADH pH 7.5, 25°C, recombinant wild-type enzyme, with L-Lys Nocardia farcinica
1.14.13.59 0.51
-
 NADPH pH 7.5, 25°C, recombinant wild-type enzyme, with D-Lys Nocardia farcinica
1.14.13.59 0.7
-
 NADPH pH 7.5, 25°C, recombinant mutant P238R, L-lysine hydroxylation Nocardia farcinica
1.14.13.59 0.8
-
 NADPH pH 7.5, 25°C, recombinant mutant P238R, O2 consumption Nocardia farcinica
1.14.13.59 1.6
-
 NADH pH 7.5, 25°C, recombinant wild-type enzyme, with D-Lys Nocardia farcinica
1.14.13.59 13
-
 L-lysine pH 7.5, 25°C, recombinant mutant K64A, with NADPH Nocardia farcinica
1.14.13.59 40
-
 L-lysine pH 7.5, 25°C, recombinant mutant K64A, with NADH Nocardia farcinica
1.14.13.59 49
-
 D-Lysine pH 7.5, 25°C, recombinant mutant K64A, with NADPH Nocardia farcinica
1.14.13.59 62
-
 D-Lysine pH 7.5, 25°C, recombinant mutant K64A, with NADH Nocardia farcinica

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.59 L-lysine + NADH + H+ + O2 Nocardia farcinica
-
 N6-hydroxy-L-lysine + NAD+ + H2O
-
 ?
1.14.13.59 L-lysine + NADPH + H+ + O2 Nocardia farcinica
-
 N6-hydroxy-L-lysine + NADP+ + H2O
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.59 Nocardia farcinica
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.59 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

EC Number Reaction Comment Organism Reaction ID
1.14.13.59 L-lysine + NADPH + H+ + O2 = N6-hydroxy-L-lysine + NADP+ + H2O via C4a-hydroperoxyflavin intermediate Nocardia farcinica
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.59 D-lysine + NADH + H+ + O2
-
 Nocardia farcinica N6-hydroxy-D-lysine + NAD+ + H2O
-
 ?
1.14.13.59 D-lysine + NADPH + H+ + O2
-
 Nocardia farcinica N6-hydroxy-D-lysine + NADP+ + H2O
-
 ?
1.14.13.59 L-lysine + NADH + H+ + O2
-
 Nocardia farcinica N6-hydroxy-L-lysine + NAD+ + H2O
-
 ?
1.14.13.59 L-lysine + NADPH + H+ + O2
-
 Nocardia farcinica N6-hydroxy-L-lysine + NADP+ + H2O
-
 ?
1.14.13.59 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

EC Number Synonyms Comment Organism
1.14.13.59 flavin-dependent lysine monooxygenase
-
 Nocardia farcinica
1.14.13.59 lysine monooxygenase
-
 Nocardia farcinica
1.14.13.59 N-hydroxylating monooxygenase
-
 Nocardia farcinica
1.14.13.59 NbtG
-
 Nocardia farcinica

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.59 25
-
 assay at Nocardia farcinica

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.13.59 0.021
-
 D-Lysine pH 7.5, 25°C, recombinant mutant K64A, with NADH Nocardia farcinica
1.14.13.59 0.13
-
 D-Lysine pH 7.5, 25°C, recombinant mutant K64A, with NADPH Nocardia farcinica
1.14.13.59 0.146
-
 L-lysine pH 7.5, 25°C, recombinant mutant P238R, with NADPH Nocardia farcinica
1.14.13.59 0.18
-
 L-lysine pH 7.5, 25°C, recombinant mutant K64A, with NADH Nocardia farcinica
1.14.13.59 0.34
-
 L-lysine pH 7.5, 25°C, recombinant mutant K64A, with NADPH Nocardia farcinica
1.14.13.59 0.6
-
 O2 pH 7.5, 25°C, recombinant mutant P238R, with NADPH Nocardia farcinica
1.14.13.59 1.08
-
 NADPH pH 7.5, 25°C, recombinant wild-type enzyme, with L-Lys Nocardia farcinica
1.14.13.59 1.29
-
 NADH pH 7.5, 25°C, recombinant wild-type enzyme, with L-Lys Nocardia farcinica
1.14.13.59 2.5
-
 NADH pH 7.5, 25°C, recombinant wild-type enzyme, with D-Lys Nocardia farcinica
1.14.13.59 2.9
-
 NADPH pH 7.5, 25°C, recombinant wild-type enzyme, with D-Lys Nocardia farcinica

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.59 7.5
-
 assay at Nocardia farcinica

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.59 FAD
-
 Nocardia farcinica
1.14.13.59 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
1.14.13.59 NADH
-
 Nocardia farcinica
1.14.13.59 NADPH
-
 Nocardia farcinica

General Information

EC Number General Information Comment Organism
1.14.13.59 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
1.14.13.59 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 [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.13.59 0.00034
-
 D-Lysine pH 7.5, 25°C, recombinant mutant K64A, with NADH Nocardia farcinica
1.14.13.59 0.0027
-
 D-Lysine pH 7.5, 25°C, recombinant mutant K64A, with NADPH Nocardia farcinica
1.14.13.59 0.0045
-
 L-lysine pH 7.5, 25°C, recombinant mutant K64A, with NADH Nocardia farcinica
1.14.13.59 0.026
-
 L-lysine pH 7.5, 25°C, recombinant mutant K64A, with NADPH Nocardia farcinica
1.14.13.59 0.21
-
 NADPH pH 7.5, 25°C, recombinant mutant P238R, L-lysine hydroxylation Nocardia farcinica
1.14.13.59 0.79
-
 NADPH pH 7.5, 25°C, recombinant mutant P238R, O2 consumption Nocardia farcinica
1.14.13.59 1.563
-
 D-Lysine pH 7.5, 25°C, recombinant wild-type enzyme, with NADH Nocardia farcinica
1.14.13.59 3.225
-
 L-lysine pH 7.5, 25°C, recombinant wild-type enzyme, with NADH Nocardia farcinica
1.14.13.59 5.686
-
 D-Lysine pH 7.5, 25°C, recombinant wild-type enzyme, with NADPH Nocardia farcinica
1.14.13.59 7.714
-
 L-lysine pH 7.5, 25°C, recombinant wild-type enzyme, with NADPH Nocardia farcinica