A flavoprotein (FAD). The enzyme from strain EN 222 of Escherichia coli is highly specific for L-lysine; L-ornithine and L-homolysine are, for example, not substrates.
unique mechanism where a rate-limiting and pH-sensitive conformational change occurs in the reductive half-reaction, which affects the efficiency of lysine hydroxylation
unique mechanism where a rate-limiting and pH-sensitive conformational change occurs in the reductive half-reaction, which affects the efficiency of lysine hydroxylation
A flavoprotein (FAD). The enzyme from strain EN 222 of Escherichia coli is highly specific for L-lysine; L-ornithine and L-homolysine are, for example, not substrates.
the enzyme displays a 3fold preference for NADPH over NADH. R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in L-Lys binding and FAD reduction
the enzyme displays a 3fold preference for NADPH over NADH. R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in L-Lys binding and FAD reduction
the enzyme displays a 3fold preference for NADPH over NADH. R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in L-Lys binding and FAD reduction
the enzyme displays a 3fold preference for NADPH over NADH. R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in L-Lys binding and FAD reduction
enzyme functions as an oxidase when the activity of MbsG is measured by monitoring oxygen consumption in the absence of L-lysine, oxidizing NADH and NADPH with kcat values of 59 and 49 per min, respectively. Under these conditions, both hydrogen peroxide and superoxide are produced
MbsG is non-specific for reduced pyridine dinucleotide, as it can utilize both NADH and NADPH with similar catalytic efficiency. But MbsG is specific for L-lysine and shows no activity with L-ornithine, 6-amino-1-hexanol, L-arginine, putrescine, and cadaverine
MbsG is non-specific for reduced pyridine dinucleotide, as it can utilize both NADH and NADPH with similar catalytic efficiency. But MbsG is specific for L-lysine and shows no activity with L-ornithine, 6-amino-1-hexanol, L-arginine, putrescine, and cadaverine
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
the enzyme displays a 3fold preference for NADPH over NADH. R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH
the enzyme displays a 3fold preference for NADPH over NADH. R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH
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
enzyme is sensitive to deleterious action of endoproteases trypsin, carboxypeptidase Y, and chymotrypsin, FAD and ADP protect, proteolysis of a C-terminal segment results in loss of enzyme activity
analysis of steady-state and rapid reaction conditions using primary and solvent kinetic isotope effects, stopped-flow and steady-state kinetics, overview
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
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
with time the enzyme aggregates to polytetrameric forms, which is reversible by thiols, the C-terminal segment is important for activity and conformational stability
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
site-directed mutagenesis, about 2fold increased activity, unaffected thermal stability, and affinity for L-lysine and FAD compared to the wild-type enzyme
activity of the genetically engineered enzyme forms C51A rIucD, C51A/C158A eIucD is 1.5times that of the parent rIucD. The activity of C158A rIucD is similar to that of the parent enzyme form
site-directed mutagenesis, about 2fold increased activity, unaffected thermal stability, and affinity for L-lysine and FAD compared to the wild-type enzyme
activity of the genetically engineered enzyme forms C51A rIucD, C51A/C158A eIucD is 1.5times that of the parent rIucD. The activity of C158A rIucD is similar to that of the parent enzyme form
site-directed mutagenesis, about 2fold increased activity, unaffected thermal stability, and affinity for L-lysine and FAD compared to the wild-type enzyme
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
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
construction of recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs result in the addition of the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involves the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms produce soluble N6-hydroxylysine
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme is sensitive to deleterious action of proteases, FAD and ADP protect, while NADPH protects only partially, and L-lysine and L-norleucine are ineffective in protecting the enzyme
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme form IucD439, in which the sequence encoding the IucD protein is fused in frame to the amino-terminal sequence of beta-galactosidase
recombinant His-tagged wild-type and P14G mutant enzymes from strain M15-2 to homogeneity by gel filtration, ion exchange and nickel affinity chromatography
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
gene aerA, DNA and amino acid sequence determination and analysis, expression of His-tagged wild-type and P14G mutant enzymes in strain M15-2, comparison of nucleotide sequences of enzyme-encoding genes iucD and aerA
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
Thariath, A.; Socha, D.; Valvano, M.A.; Viswanatha, T.
Construction and biochemical characterization of recombinant cytoplasmic forms of IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster
Substrate binding modulates the activity of Mycobacterium smegmatis G, a flavin-dependent monooxygenase involved in the biosynthesis of hydroxamate-containing siderophores
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