Cloned (Comment) | Organism |
---|---|
gene sidA, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)-T1 | Aspergillus fumigatus |
Crystallization (Comment) | Organism |
---|---|
purified recombinant detagged enzyme mutant N323A complexed with NADP+ and ornithine, hanging drop vapor diffusion method and microseeding, mixing of mixing of 8 mg/ml SidA N323A mutant protein, preincubated with 1 mM NADP+ and 100 mM ornithine, with an equal volume of reservoir solution containing 0.1 M HEPES, pH 6.6, 1.86 M ammonium sulfate, and 1% v/v dioxane, X-ray diffraction structure determination and analysis at 2.10 A resolution | Aspergillus fumigatus |
Protein Variants | Comment | Organism |
---|---|---|
K107A | site-directed mutagenesis, inactive mutant | Aspergillus fumigatus |
N293A | site-directed mutagenesis, the mutation leads to highly increased Km for L-ornithine compared to the wild-type | Aspergillus fumigatus |
N323A | site-directed mutagenesis, the mutation leads to highly increased Km for L-ornithine compared to the wild-type, and to increased rate constant for flavin reduction by NADPH by 18fold | Aspergillus fumigatus |
S469A | site-directed mutagenesis, the mutation leads to highly increased Km for L-ornithine compared to the wild-type | Aspergillus fumigatus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Pre-steady-state kinetics and steady-state kinetics | Aspergillus fumigatus | |
0.007 | - |
NADPH | pH 7.5, 25°C, recombinant wild-type enzyme, O2 consumption assay | Aspergillus fumigatus | |
0.01 | - |
NADPH | pH 7.5, 25°C, recombinant mutant N323A, O2 consumption assay | Aspergillus fumigatus | |
0.025 | - |
NADPH | pH 7.5, 25°C, recombinant mutant S469A, O2 consumption assay | Aspergillus fumigatus | |
0.06 | - |
NADPH | pH 7.5, 25°C, recombinant mutant N293A, O2 consumption assay | Aspergillus fumigatus | |
1 | - |
L-ornithine | pH 7.5, 25°C, recombinant wild-type enzyme, ornithine hydroxylation assay | Aspergillus fumigatus | |
1.1 | - |
L-ornithine | pH 7.5, 25°C, recombinant wild-type enzyme, O2 consumption assay | Aspergillus fumigatus | |
12 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant N323A, ornithine hydroxylation assay | Aspergillus fumigatus | |
15 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant N323A, O2 consumption assay | Aspergillus fumigatus | |
16 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant N293A, ornithine hydroxylation assay | Aspergillus fumigatus | |
18 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant N293A, O2 consumption assay | Aspergillus fumigatus | |
18 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant S469A, ornithine hydroxylation assay | Aspergillus fumigatus | |
21 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant S469A, O2 consumption assay | Aspergillus fumigatus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-ornithine + NADPH + H+ + O2 | Aspergillus fumigatus | - |
N5-hydroxy-L-ornithine + NADP+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Aspergillus fumigatus | E9QYP0 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)-T1 by affinity chromatography, proteolytic tag removal | Aspergillus fumigatus |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
L-ornithine + NADPH + H+ + O2 = N5-hydroxy-L-ornithine + NADP+ + H2O | sequential kinetic mechanism for SidA. The reaction is initiated by binding of NADPH to SidA with the flavin in its oxidized form (FADox). The rate-limiting step in the reaction is the stereospecific transfer of the pro-R-hydride equivalent from NADPH to yield reduced flavin (FADred) and NADP+. The FADred-NADP+ complex reacts with molecular oxygen forming the C4a-hydroperoxyflavin (FADOOH). In this complex, NADP+ is essential for stabilization of the FADOOH. After ornithine (Orn) binding, hydroxylation occurs very quickly. The last step in the reaction is the release of products | Aspergillus fumigatus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-ornithine + NADPH + H+ + O2 | - |
Aspergillus fumigatus | N5-hydroxy-L-ornithine + NADP+ + H2O | - |
? |
Synonyms | Comment | Organism |
---|---|---|
ornithine N5-monooxygenase | - |
Aspergillus fumigatus |
SidA | - |
Aspergillus fumigatus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Aspergillus fumigatus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.11 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant S469A, ornithine hydroxylation assay | Aspergillus fumigatus | |
0.15 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant S469A, O2 consumption assay | Aspergillus fumigatus | |
0.5 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant N323A, ornithine hydroxylation assay | Aspergillus fumigatus | |
0.53 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant N293A, ornithine hydroxylation assay | Aspergillus fumigatus | |
0.59 | - |
L-ornithine | pH 7.5, 25°C, recombinant wild-type enzyme, O2 consumption assay | Aspergillus fumigatus | |
0.62 | - |
L-ornithine | pH 7.5, 25°C, recombinant wild-type enzyme, ornithine hydroxylation assay | Aspergillus fumigatus | |
0.88 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant N293A, O2 consumption assay | Aspergillus fumigatus | |
1.06 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant N323A, O2 consumption assay | Aspergillus fumigatus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Aspergillus fumigatus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADPH | dependent on | Aspergillus fumigatus |
General Information | Comment | Organism |
---|---|---|
additional information | residue Asn323 interacts with the enzyme and also interacts with NADPH by forming a hydrogen bond with the nicotinamide ribose, residue K107 is important for catalytic activity. Asn323 thus facilitates ornithine binding at the expense of hindering flavin reduction | Aspergillus fumigatus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.006 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant S469A, O2 consumption assay | Aspergillus fumigatus | |
0.006 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant S469A, ornithine hydroxylation assay | Aspergillus fumigatus | |
0.033 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant N293A, O2 consumption assay | Aspergillus fumigatus | |
0.033 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant N293A, ornithine hydroxylation assay | Aspergillus fumigatus | |
0.042 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant N323A, O2 consumption assay | Aspergillus fumigatus | |
0.042 | - |
L-ornithine | pH 7.5, 25°C, recombinant mutant N323A, ornithine hydroxylation assay | Aspergillus fumigatus | |
0.62 | - |
L-ornithine | pH 7.5, 25°C, recombinant wild-type enzyme, O2 consumption assay | Aspergillus fumigatus | |
0.62 | - |
L-ornithine | pH 7.5, 25°C, recombinant wild-type enzyme, ornithine hydroxylation assay | Aspergillus fumigatus | |
6 | - |
NADPH | pH 7.5, 25°C, recombinant mutant S469A, O2 consumption assay | Aspergillus fumigatus | |
15 | - |
NADPH | pH 7.5, 25°C, recombinant mutant N293A, O2 consumption assay | Aspergillus fumigatus | |
84.3 | - |
NADPH | pH 7.5, 25°C, recombinant wild-type enzyme, O2 consumption assay | Aspergillus fumigatus | |
110 | - |
NADPH | pH 7.5, 25°C, recombinant mutant N323A, O2 consumption assay | Aspergillus fumigatus |