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Literature summary extracted from

  • Robinson, R.; Qureshi, I.A.; Klancher, C.A.; Rodriguez, P.J.; Tanner, J.J.; Sobrado, P.
    Contribution to catalysis of ornithine binding residues in ornithine N5-monooxygenase (2015), Arch. Biochem. Biophys., 585, 25-31 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.195 gene sidA, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)-T1 Aspergillus fumigatus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.14.13.195 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

EC Number Protein Variants Comment Organism
1.14.13.195 K107A site-directed mutagenesis, inactive mutant Aspergillus fumigatus
1.14.13.195 N293A site-directed mutagenesis, the mutation leads to highly increased Km for L-ornithine compared to the wild-type Aspergillus fumigatus
1.14.13.195 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
1.14.13.195 S469A site-directed mutagenesis, the mutation leads to highly increased Km for L-ornithine compared to the wild-type Aspergillus fumigatus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.195 additional information
-
 additional information Pre-steady-state kinetics and steady-state kinetics Aspergillus fumigatus
1.14.13.195 0.007
-
 NADPH pH 7.5, 25°C, recombinant wild-type enzyme, O2 consumption assay Aspergillus fumigatus
1.14.13.195 0.01
-
 NADPH pH 7.5, 25°C, recombinant mutant N323A, O2 consumption assay Aspergillus fumigatus
1.14.13.195 0.025
-
 NADPH pH 7.5, 25°C, recombinant mutant S469A, O2 consumption assay Aspergillus fumigatus
1.14.13.195 0.06
-
 NADPH pH 7.5, 25°C, recombinant mutant N293A, O2 consumption assay Aspergillus fumigatus
1.14.13.195 1
-
 L-ornithine pH 7.5, 25°C, recombinant wild-type enzyme, ornithine hydroxylation assay Aspergillus fumigatus
1.14.13.195 1.1
-
 L-ornithine pH 7.5, 25°C, recombinant wild-type enzyme, O2 consumption assay Aspergillus fumigatus
1.14.13.195 12
-
 L-ornithine pH 7.5, 25°C, recombinant mutant N323A, ornithine hydroxylation assay Aspergillus fumigatus
1.14.13.195 15
-
 L-ornithine pH 7.5, 25°C, recombinant mutant N323A, O2 consumption assay Aspergillus fumigatus
1.14.13.195 16
-
 L-ornithine pH 7.5, 25°C, recombinant mutant N293A, ornithine hydroxylation assay Aspergillus fumigatus
1.14.13.195 18
-
 L-ornithine pH 7.5, 25°C, recombinant mutant N293A, O2 consumption assay Aspergillus fumigatus
1.14.13.195 18
-
 L-ornithine pH 7.5, 25°C, recombinant mutant S469A, ornithine hydroxylation assay Aspergillus fumigatus
1.14.13.195 21
-
 L-ornithine pH 7.5, 25°C, recombinant mutant S469A, O2 consumption assay Aspergillus fumigatus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.195 L-ornithine + NADPH + H+ + O2 Aspergillus fumigatus
-
 N5-hydroxy-L-ornithine + NADP+ + H2O
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.195 Aspergillus fumigatus E9QYP0
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.195 recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)-T1 by affinity chromatography, proteolytic tag removal Aspergillus fumigatus

Reaction

EC Number Reaction Comment Organism Reaction ID
1.14.13.195 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
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.195 L-ornithine + NADPH + H+ + O2
-
 Aspergillus fumigatus N5-hydroxy-L-ornithine + NADP+ + H2O
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.14.13.195 ornithine N5-monooxygenase
-
 Aspergillus fumigatus
1.14.13.195 SidA
-
 Aspergillus fumigatus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.195 25
-
 assay at Aspergillus fumigatus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.13.195 0.11
-
 L-ornithine pH 7.5, 25°C, recombinant mutant S469A, ornithine hydroxylation assay Aspergillus fumigatus
1.14.13.195 0.15
-
 L-ornithine pH 7.5, 25°C, recombinant mutant S469A, O2 consumption assay Aspergillus fumigatus
1.14.13.195 0.5
-
 L-ornithine pH 7.5, 25°C, recombinant mutant N323A, ornithine hydroxylation assay Aspergillus fumigatus
1.14.13.195 0.53
-
 L-ornithine pH 7.5, 25°C, recombinant mutant N293A, ornithine hydroxylation assay Aspergillus fumigatus
1.14.13.195 0.59
-
 L-ornithine pH 7.5, 25°C, recombinant wild-type enzyme, O2 consumption assay Aspergillus fumigatus
1.14.13.195 0.62
-
 L-ornithine pH 7.5, 25°C, recombinant wild-type enzyme, ornithine hydroxylation assay Aspergillus fumigatus
1.14.13.195 0.88
-
 L-ornithine pH 7.5, 25°C, recombinant mutant N293A, O2 consumption assay Aspergillus fumigatus
1.14.13.195 1.06
-
 L-ornithine pH 7.5, 25°C, recombinant mutant N323A, O2 consumption assay Aspergillus fumigatus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.195 7.5
-
 assay at Aspergillus fumigatus

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.195 NADPH dependent on Aspergillus fumigatus

General Information

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

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.13.195 0.006
-
 L-ornithine pH 7.5, 25°C, recombinant mutant S469A, O2 consumption assay Aspergillus fumigatus
1.14.13.195 0.006
-
 L-ornithine pH 7.5, 25°C, recombinant mutant S469A, ornithine hydroxylation assay Aspergillus fumigatus
1.14.13.195 0.033
-
 L-ornithine pH 7.5, 25°C, recombinant mutant N293A, O2 consumption assay Aspergillus fumigatus
1.14.13.195 0.033
-
 L-ornithine pH 7.5, 25°C, recombinant mutant N293A, ornithine hydroxylation assay Aspergillus fumigatus
1.14.13.195 0.042
-
 L-ornithine pH 7.5, 25°C, recombinant mutant N323A, O2 consumption assay Aspergillus fumigatus
1.14.13.195 0.042
-
 L-ornithine pH 7.5, 25°C, recombinant mutant N323A, ornithine hydroxylation assay Aspergillus fumigatus
1.14.13.195 0.62
-
 L-ornithine pH 7.5, 25°C, recombinant wild-type enzyme, O2 consumption assay Aspergillus fumigatus
1.14.13.195 0.62
-
 L-ornithine pH 7.5, 25°C, recombinant wild-type enzyme, ornithine hydroxylation assay Aspergillus fumigatus
1.14.13.195 6
-
 NADPH pH 7.5, 25°C, recombinant mutant S469A, O2 consumption assay Aspergillus fumigatus
1.14.13.195 15
-
 NADPH pH 7.5, 25°C, recombinant mutant N293A, O2 consumption assay Aspergillus fumigatus
1.14.13.195 84.3
-
 NADPH pH 7.5, 25°C, recombinant wild-type enzyme, O2 consumption assay Aspergillus fumigatus
1.14.13.195 110
-
 NADPH pH 7.5, 25°C, recombinant mutant N323A, O2 consumption assay Aspergillus fumigatus