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EC Tree
IUBMB Comments A flavoprotein (FAD). The enzyme from the pathogenic fungus Aspergillus fumigatus catalyses a step in the biosynthesis of the siderophores triacetylfusarinine and desferriferricrocin, while the enzyme from the bacterium Kutzneria sp. 744 is involved in the biosynthesis of piperazate, a building block of the kutzneride family of antifungal antibiotics. Activity of the fungal enzyme is higher with NADPH, due to the fact that following the reduction of the flavin, NADP+ (but not NAD+) stabilizes the C4a-hydroperoxyflavin intermediate that oxidizes the substrate . cf. EC 1.14.13.195, L-ornithine N5-monooxygenase (NADPH).
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
Synonyms
abachelin monooxygenase, AfSidA, AMO, SidA, siderophore A,
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abachelin monooxygenase
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SidA
Q5SE95
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L-ornithine + NAD(P)H + H+ + O2 = N5-hydroxy-L-ornithine + NAD(P)+ + H2O
L-ornithine + NAD(P)H + H+ + O2 = N5-hydroxy-L-ornithine + NAD(P)+ + H2O
the enzyme forms a ternary complex with NADP and L-ornithine during catalysis. The enzyme likely proceeds by a sequential kinetic mechanism
Q5SE95
L-ornithine + NAD(P)H + H+ + O2 = N5-hydroxy-L-ornithine + NAD(P)+ + H2O
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L-ornithine,NAD(P)H:oxygen oxidoreductase (N5-hydroxylating)
A flavoprotein (FAD). The enzyme from the pathogenic fungus Aspergillus fumigatus catalyses a step in the biosynthesis of the siderophores triacetylfusarinine and desferriferricrocin, while the enzyme from the bacterium Kutzneria sp. 744 is involved in the biosynthesis of piperazate, a building block of the kutzneride family of antifungal antibiotics. Activity of the fungal enzyme is higher with NADPH, due to the fact that following the reduction of the flavin, NADP+ (but not NAD+) stabilizes the C4a-hydroperoxyflavin intermediate that oxidizes the substrate [3]. cf. EC 1.14.13.195, L-ornithine N5-monooxygenase (NADPH).
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L-ornithine + NAD(P)H + H+ + O2
N5-hydroxy-L-ornithine + NAD(P)+ + H2O
Q5SE95
the enzyme catalyzes the hydroxylation of L-ornithine in ferrichrome biosynthesis
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L-ornithine + NADH + H+ + O2
N5-hydroxy-L-ornithine + NAD+ + H2O
L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
additional information
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holo (FAD-bound) and apo (FAD-free) forms of the enzyme hydroxylate ornithine. No activity with L-lysine. The enzyme displays very low selectivity for the reduced nicotinamide coenzyme
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L-ornithine + NADH + H+ + O2
N5-hydroxy-L-ornithine + NAD+ + H2O
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L-ornithine + NADH + H+ + O2
N5-hydroxy-L-ornithine + NAD+ + H2O
Q5SE95
first step in the biosynthesis of hydroxamate-containing siderophores, essential for virulence
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L-ornithine + NADH + H+ + O2
N5-hydroxy-L-ornithine + NAD+ + H2O
Q5SE95
the enzyme catalyzes the generation of N5-hydroxyornithine in the biosynthesis of siderophores, a reaction essential for virulence
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L-ornithine + NADH + H+ + O2
N5-hydroxy-L-ornithine + NAD+ + H2O
Q5SE95
the enzyme is essential for virulence in Aspergillus fumigatus
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L-ornithine + NADH + H+ + O2
N5-hydroxy-L-ornithine + NAD+ + H2O
Q5SE95
accepts reducing equivalents from either NADPH or NADH and displays similar kinetic parameters when using either coenzyme
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L-ornithine + NADH + H+ + O2
N5-hydroxy-L-ornithine + NAD+ + H2O
Q5SE95
AfSidA is highly specific for its amino acid substrate, only hydroxylating L-ornithine. An 8-fold preference in the catalytic efficiency is determined for NADPH compared to NADH
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
Q5SE95
first step in the biosynthesis of hydroxamate-containing siderophores, essential for virulence
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
Q5SE95
the enzyme catalyzes the generation of N5-hydroxyornithine in the biosynthesis of siderophores, a reaction essential for virulence
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
Q5SE95
the enzyme catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
Q5SE95
the enzyme is essential for virulence in Aspergillus fumigatus
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
Q5SE95
accepts reducing equivalents from either NADPH or NADH and displays similar kinetic parameters when using either coenzyme
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
Q5SE95
AfSidA is highly specific for its amino acid substrate, only hydroxylating L-ornithine. An 8-fold preference in the catalytic efficiency is determined for NADPH compared to NADH
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
Q5SE95
in the reductive half-reaction, the oxidized FAD bound to the enzyme, is reduced by NADPH. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
Q5SE95
NADPH reduces the flavin, and the resulting NADP+ is the last product to be released
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L-ornithine + NAD(P)H + H+ + O2
N5-hydroxy-L-ornithine + NAD(P)+ + H2O
Q5SE95
the enzyme catalyzes the hydroxylation of L-ornithine in ferrichrome biosynthesis
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L-ornithine + NADH + H+ + O2
N5-hydroxy-L-ornithine + NAD+ + H2O
L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
L-ornithine + NADH + H+ + O2
N5-hydroxy-L-ornithine + NAD+ + H2O
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L-ornithine + NADH + H+ + O2
N5-hydroxy-L-ornithine + NAD+ + H2O
Q5SE95
first step in the biosynthesis of hydroxamate-containing siderophores, essential for virulence
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L-ornithine + NADH + H+ + O2
N5-hydroxy-L-ornithine + NAD+ + H2O
Q5SE95
the enzyme catalyzes the generation of N5-hydroxyornithine in the biosynthesis of siderophores, a reaction essential for virulence
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L-ornithine + NADH + H+ + O2
N5-hydroxy-L-ornithine + NAD+ + H2O
Q5SE95
the enzyme is essential for virulence in Aspergillus fumigatus
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
Q5SE95
first step in the biosynthesis of hydroxamate-containing siderophores, essential for virulence
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?
L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
Q5SE95
the enzyme catalyzes the generation of N5-hydroxyornithine in the biosynthesis of siderophores, a reaction essential for virulence
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
Q5SE95
the enzyme catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence
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L-ornithine + NADPH + H+ + O2
N5-hydroxy-L-ornithine + NADP+ + H2O
Q5SE95
the enzyme is essential for virulence in Aspergillus fumigatus
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FAD
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FAD
Q5SE95
FAD-containing enzyme. In the reductive half-reaction, the oxidized FAD bound to the enzyme, is reduced by NADPH. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine
NADH
Q5SE95
8-fold preference in the catalytic efficiency is determined for NADPH compared to NADH
NADH
Q5SE95
accepts reducing equivalents from either NADPH or NADH and displays similar kinetic parameters when using either coenzyme
NADH
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the enzyme displays very low selectivity for the reduced nicotinamide coenzyme
NADPH
Q5SE95
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NADPH
Q5SE95
8-fold preference in the catalytic efficiency is determined for NADPH compared to NADH
NADPH
Q5SE95
accepts reducing equivalents from either NADPH or NADH and displays similar kinetic parameters when using either coenzyme. NADP+, and not NAD+, protects the enzyme from proteolysis
NADPH
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the enzyme displays very low selectivity for the reduced nicotinamide coenzyme
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NADH
Q5SE95
substrate inhibition
NADP+
Q5SE95
competitive inhibitor with respect to NADPH
NADPH
Q5SE95
substrate inhibition
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additional information
additional information
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steady-state kinetic analysis
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0.107
L-ornithine
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recombinant FAD-reconstituted enzyme-enzyme, pH 7.5, temperature not specified in the publication
0.21
L-ornithine
Q5SE95
pH 7.6, 25°C, mutant enzyme S257A, cofactor: NADPH
0.3
L-ornithine
Q5SE95
pH 7.6, 25°C, wild-type enzyme, cofactor: NADPH
0.305
L-ornithine
-
recombinant holo-enzyme, pH 7.5, temperature not specified in the publication
0.5
L-ornithine
Q5SE95
pH 7.5, 25°C, cosubstrate: NADH, oxygen consumption assay
1.7
L-ornithine
Q5SE95
pH 7.5, 25°C, cosubstrate: NADH, following the formation of hydroxylated ornithine
1.7
L-ornithine
Q5SE95
pH 7.5, 25°C, cosubstrate: NADPH, following the formation of hydroxylated ornithine
2
L-ornithine
Q5SE95
pH 7.5, 25°C, cosubstrate: NADPH, oxygen consumption assay
0.1
NADH
Q5SE95
pH 7.5, temperature not specified in the publication
0.21
NADH
Q5SE95
pH 7.5, 25°C, oxygen consumption assay
0.94
NADH
Q5SE95
pH 7.5, 25°C, following the formation of hydroxylated ornithine
0.012
NADPH
Q5SE95
pH 7.5, temperature not specified in the publication
0.014
NADPH
Q5SE95
pH 7.6, 25°C, mutant enzyme S257A
0.017
NADPH
Q5SE95
pH 7.6, 25°C, wild-type enzyme
0.03
NADPH
Q5SE95
pH 7.5, 25°C, oxygen consumption assay
0.9
NADPH
Q5SE95
pH 7.5, 25°C, following the formation of hydroxylated ornithine
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additional information
additional information
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0.03
L-ornithine
Q5SE95
pH 7.6, 25°C, mutant enzyme S257A, cofactor: NADPH
0.03
L-ornithine
Q5SE95
pH 7.6, 25°C, wild-type enzyme, cofactor: NADPH
0.2
L-ornithine
-
recombinant FAD-reconstituted enzyme-enzyme, pH 7.5, temperature not specified in the publication
0.34
L-ornithine
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recombinant holo-enzyme, pH 7.5, temperature not specified in the publication
0.48
L-ornithine
Q5SE95
pH 7.5, 25°C, cosubstrate: NADPH, following the formation of hydroxylated ornithine
0.5
L-ornithine
Q5SE95
pH 7.5, 25°C, cosubstrate: NADH, following the formation of hydroxylated ornithine
0.68
L-ornithine
Q5SE95
pH 7.5, 25°C, cosubstrate: NADH, oxygen consumption assay
0.87
L-ornithine
Q5SE95
pH 7.5, 25°C, cosubstrate: NADPH, oxygen consumption assay
0.73
NADH
Q5SE95
pH 7.5, temperature not specified in the publication
1.18
NADH
Q5SE95
pH 7.5, 25°C, following the formation of hydroxylated ornithine
1.36
NADH
Q5SE95
pH 7.5, 25°C, oxygen consumption assay
0.6
NADPH
Q5SE95
pH 7.6, 25°C, wild-type enzyme
0.6
NADPH
Q5SE95
pH 7.5, temperature not specified in the publication
0.63
NADPH
Q5SE95
pH 7.5, 25°C, oxygen consumption assay
0.85
NADPH
Q5SE95
pH 7.6, 25°C, mutant enzyme S257A
1.25
NADPH
Q5SE95
pH 7.5, 25°C, following the formation of hydroxylated ornithine
additional information
additional information
Q5SE95
monitoring the reductive and oxidative half-reactions of a flavin-dependent monooxygenase using stopped-flow spectrophotometry
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additional information
additional information
Q5SE95
presteady-state kinetic parameters, cofactor: NADPH
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0.09
L-ornithine
Q5SE95
pH 7.6, 25°C, mutant enzyme S257A, cofactor: NADPH
0.14
L-ornithine
Q5SE95
pH 7.6, 25°C, wild-type enzyme, cofactor: NADPH
0.28
L-ornithine
Q5SE95
pH 7.5, 25°C, cosubstrate: NADH, following the formation of hydroxylated ornithine
0.29
L-ornithine
Q5SE95
pH 7.5, 25°C, cosubstrate: NADPH, following the formation of hydroxylated ornithine
0.43
L-ornithine
Q5SE95
pH 7.5, 25°C, cosubstrate: NADH, oxygen consumption assay
1.12
L-ornithine
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recombinant holo-enzyme, pH 7.5, temperature not specified in the publication
1.33
L-ornithine
Q5SE95
pH 7.5, 25°C, cosubstrate: NADPH, oxygen consumption assay
1.7
L-ornithine
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recombinant FAD-reconstituted enzyme-enzyme, pH 7.5, temperature not specified in the publication
1.3
NADH
Q5SE95
pH 7.5, 25°C, following the formation of hydroxylated ornithine
3.4
NADH
Q5SE95
pH 7.5, 25°C, oxygen consumption assay
7.3
NADH
Q5SE95
pH 7.5, temperature not specified in the publication
1.3
NADPH
Q5SE95
pH 7.5, 25°C, following the formation of hydroxylated ornithine
14
NADPH
Q5SE95
pH 7.6, 25°C, wild-type enzyme
21.05
NADPH
Q5SE95
pH 7.5, 25°C, oxygen consumption assay
40
NADPH
Q5SE95
pH 7.6, 25°C, mutant enzyme S257A
50
NADPH
Q5SE95
pH 7.5, temperature not specified in the publication
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1.9
NADH
Q5SE95
pH 7.5, 25°C
2.8
NADPH
Q5SE95
pH 7.5, 25°C
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7.5
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assay at
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7.3
calculated from sequence
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brenda
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Q5SE95
UniProt
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UniProt
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UniProt
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UniProt
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additional information
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Amycolatopsis alba cells grown under iron-limiting conditions
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evolution
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AMO is a member of the N-hydroxylating monooxygenase (NMO) family of enzymes
metabolism
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the enzyme is an N-hydroxylating monooxygenase (NMO) catalyzing the ornithine hydroxylation step in albachelin biosynthesis
physiological function
Q5SE95
first step in the biosynthesis of hydroxamate-containing siderophores, essential for virulence
physiological function
Q5SE95
the enzyme is essential for virulence in Aspergillus fumigatus
physiological function
mutations in sidA abolish both epichloenin A and ferricrocin production. Mutants form colonies that are viable but display an iron starvation phenotype
physiological function
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mutations in sidA abolish both epichloenin A and ferricrocin production. Mutants form colonies that are viable but display an iron starvation phenotype
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228000
Q5SE95
gel filtration
55000
Q5SE95
4 * 55000, SDS-PAGE
57210
Q5SE95
4 * 57210, MALDI-TOF MS
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?
x * 55200, calculated from sequence
tetramer
Q5SE95
4 * 55000, SDS-PAGE
tetramer
Q5SE95
4 * 57210, MALDI-TOF MS
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glycoprotein
sequence contains three possible N-linked glycosylation sites
glycoprotein
sequence contains three putative N-linked glycosylation sites
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crystal structure of the enzyme bound to NADP+ and L-ornithine (1.9 A resolution), crystal structure of the enzyme bound to NADP+ and L-lysine (2.28 A resolution), crystal structure of the enzyme (reduced state) bound to NADPH (2.32 A resolution), crystal structure of the enzyme (reduced state) bound to NADP+ and L-arginine (2.9 A resolution), crystal structure of the enzyme (re-oxidized state) bound to NADP+ and L-ornithine (2.75 A resolution), crystal structure of the enzyme (re-oxidized state) bound to NADP+ and L-arginine (2.29 A resolution), crystal structure of the enzyme bound to L-ornithine (2.3 A resolution)
Q5SE95
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S257A
Q5SE95
following oxygen consumption, the Km value of the S257A enzyme for NADPH was not significantly changed, whereas the kcat value increased. When the activity is determined by measuring the formation of N5-hydroxyornithine, the kcat value for the S257A enzyme is lower than that for the wild-type enzyme. The S257A mutation causes an increase in flexibility in two small loops, loop 253257, which is within interacting distance of NADP+ (including Ser257 and Gln256, which are able to form hydrogen bonds with NADP during MD simulations), and loop 453458, which resides 5 A from the FAD phosphate group. In contrast, loop 97105 (including Gln-102, which makes a hydrogen bond with FAD) presents reduced flexibility in the mutant enzyme
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NADP+, and not NAD+, protects the enzyme from proteolysis
Q5SE95
the presence of NADP+ is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species
Q5SE95
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recombinant His-tagged enzyme from Escherichia coli by metal affinity chromatography, depending on the imidazole concentration used, the recombinant enzyme is isolated either in the apo (FAD-free) or holo (FAD-bound) form, cleavage of the tag by TEV protease
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expressed as a fusion to MBP containing an 8His tag at the N-terminus in Escherichia coli BL21T1R cells
Q5SE95
expression in Escherichia coli
Q5SE95
recombinant expression of N-terminally His8-MBP-tagged enzyme in Escherichia coli as soluble protein
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drug development
Q5SE95
essential for virulence, validating this enzyme as a drug target
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Chocklett, S.W.; Sobrado, P.
Aspergillus fumigatus SidA is a highly specific ornithine hydroxylase with bound flavin cofactor
Biochemistry
49
6777-6783
2010
Aspergillus fumigatus (Q5SE95)
brenda
Franceschini, S.; Fedkenheuer, M.; Vogelaar, N.J.; Robinson, H.H.; Sobrado, P.; Mattevi, A..
Structural insight into the mechanism of oxygen activation and substrate selectivity of flavin-dependent N-hydroxylating monooxygenases
Biochemistry
51
7043-7045
2012
Aspergillus fumigatus (Q5SE95)
brenda
Romero, E.; Fedkenheuer, M.; Chocklett, S,W.; Qi, J.; Oppenheimer, M.; Sobrado, P.
Dual role of NADP(H) in the reaction of a flavin dependent N-hydroxylating monooxygenase
Biochim. Biophys. Acta
1824
850-857
2012
Aspergillus fumigatus (Q5SE95)
brenda
Shirey, C.; Badieyan, S.; Sobrado, P.
Role of Ser-257 in the sliding mechanism of NADP(H) in the reaction catalyzed by the Aspergillus fumigatus flavin-dependent ornithine N5-monooxygenase SidA
J. Biol. Chem.
288
32440-32448
2013
Aspergillus fumigatus (Q5SE95)
brenda
Romero, E.; Robinson, R.; Sobrado, P.
Monitoring the reductive and oxidative half-reactions of a flavin-dependent monooxygenase using stopped-flow spectrophotometry
J. Vis. Exp.
18
pii: 3803
2012
Aspergillus fumigatus (Q5SE95)
brenda
Bufkin, K.; Sobrado, P.
Characterization of the ornithine hydroxylation step in albachelin biosynthesis
Molecules
22
E1652
2017
Amycolatopsis alba
brenda
Kitiyanant, V.; Kanchanabanca, C.; Punnapayak, H.; Satjarak, A.; Lotrakul, P.; Prasongsuk, S.
The sida gene of aureobasidium thailandense and its phylogenetic relationship among those of Aureobasidium species
Chiang Mai J. Sci.
48
13-26
2021
Aureobasidium thailandense (A0A6M9BS13)
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brenda
Forester, N.; Lane, G.; Steringa, M.; Lamont, I.; Johnson, L.
Contrasting roles of fungal siderophores in maintaining iron homeostasis in Epichloe festucae
Fungal Genet. Biol.
111
60-72
2018
Epichloe festucae (A0A7U3Q030), Epichloe festucae Fl1 (A0A7U3Q030)
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