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Literature summary extracted from

  • 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 (2013), J. Biol. Chem., 288, 32440-32448.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.195
-
 Aspergillus fumigatus
1.14.13.196 expression in Escherichia coli Aspergillus fumigatus

Protein Variants

EC Number Protein Variants Comment Organism
1.14.13.195 S257A the interaction between residue Ser257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin intermediate. Ser257 may function as a pivot point, allowing the nicotinamide of NADP+ to slide into position for stabilization of the C4a-hydroperoxyflavin Aspergillus fumigatus
1.14.13.196 S257A 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 253–257, 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 453–458, which resides 5 A from the FAD phosphate group. In contrast, loop 97–105 (including Gln-102, which makes a hydrogen bond with FAD) presents reduced flexibility in the mutant enzyme Aspergillus fumigatus

General Stability

EC Number General Stability Organism
1.14.13.196 the presence of NADP+ is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species Aspergillus fumigatus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.195 0.014
-
 NADPH mutant S257A, O2 consumption assay, pH 7.5, 25°C Aspergillus fumigatus
1.14.13.195 0.017
-
 NADPH wild-type, O2 consumption assay, pH 7.5, 25°C Aspergillus fumigatus
1.14.13.195 0.34
-
 NADPH mutant S257A, hydroxylamine detection assay, pH 7.5, 25°C Aspergillus fumigatus
1.14.13.195 0.8
-
 NADPH wild-type, hydroxylamine detection assay, pH 7.5, 25°C Aspergillus fumigatus
1.14.13.196 0.014
-
 NADPH pH 7.6, 25°C, mutant enzyme S257A Aspergillus fumigatus
1.14.13.196 0.017
-
 NADPH pH 7.6, 25°C, wild-type enzyme Aspergillus fumigatus
1.14.13.196 0.21
-
 L-ornithine pH 7.6, 25°C, mutant enzyme S257A, cofactor: NADPH Aspergillus fumigatus
1.14.13.196 0.3
-
 L-ornithine pH 7.6, 25°C, wild-type enzyme, cofactor: NADPH Aspergillus fumigatus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.196 L-ornithine + NADH + H+ + O2 Aspergillus fumigatus the enzyme is essential for virulence in Aspergillus fumigatus N5-hydroxy-L-ornithine + NAD+ + H2O
-
 ?
1.14.13.196 L-ornithine + NADPH + H+ + O2 Aspergillus fumigatus the enzyme is essential for virulence in Aspergillus fumigatus N5-hydroxy-L-ornithine + NADP+ + H2O
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.195 Aspergillus fumigatus Q5SE95
-
-
1.14.13.196 Aspergillus fumigatus Q5SE95
-
-

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
-
 ?
1.14.13.196 L-ornithine + NADH + H+ + O2 the enzyme is essential for virulence in Aspergillus fumigatus Aspergillus fumigatus N5-hydroxy-L-ornithine + NAD+ + H2O
-
 ?
1.14.13.196 L-ornithine + NADPH + H+ + O2 the enzyme is essential for virulence in Aspergillus fumigatus Aspergillus fumigatus N5-hydroxy-L-ornithine + NADP+ + H2O
-
 ?
1.14.13.196 L-ornithine + NADPH + H+ + O2 NADPH reduces the flavin, and the resulting NADP+ is the last product to be released Aspergillus fumigatus N5-hydroxy-L-ornithine + NADP+ + H2O
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.14.13.196 SidA
-
 Aspergillus fumigatus
1.14.13.196 siderophore A
-
 Aspergillus fumigatus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.196 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 0.12
-
 NADPH mutant S257A, hydroxylamine detection assay, pH 7.5, 25°C Aspergillus fumigatus
1.14.13.195 0.45
-
 NADPH wild-type, hydroxylamine detection assay, pH 7.5, 25°C Aspergillus fumigatus
1.14.13.195 0.6
-
 NADPH wild-type, O2 consumption assay, pH 7.5, 25°C Aspergillus fumigatus
1.14.13.195 0.85
-
 NADPH mutant S257A, O2 consumption assay, pH 7.5, 25°C Aspergillus fumigatus
1.14.13.196 additional information
-
 additional information presteady-state kinetic parameters, cofactor: NADPH Aspergillus fumigatus
1.14.13.196 0.03
-
 L-ornithine pH 7.6, 25°C, mutant enzyme S257A, cofactor: NADPH Aspergillus fumigatus
1.14.13.196 0.03
-
 L-ornithine pH 7.6, 25°C, wild-type enzyme, cofactor: NADPH Aspergillus fumigatus
1.14.13.196 0.6
-
 NADPH pH 7.6, 25°C, wild-type enzyme Aspergillus fumigatus
1.14.13.196 0.85
-
 NADPH pH 7.6, 25°C, mutant enzyme S257A Aspergillus fumigatus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.196 7.5
-
 assay at Aspergillus fumigatus

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.196 NADPH
-
 Aspergillus fumigatus

General Information

EC Number General Information Comment Organism
1.14.13.196 physiological function the enzyme is essential for virulence in Aspergillus fumigatus 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 0.34
-
 NADPH mutant S257A, hydroxylamine detection assay, pH 7.5, 25°C Aspergillus fumigatus
1.14.13.195 0.54
-
 NADPH wild-type, hydroxylamine detection assay, pH 7.5, 25°C Aspergillus fumigatus
1.14.13.195 14
-
 NADPH wild-type, O2 consumption assay, pH 7.5, 25°C Aspergillus fumigatus
1.14.13.195 40
-
 NADPH mutant S257A, O2 consumption assay, pH 7.5, 25°C Aspergillus fumigatus
1.14.13.196 0.09
-
 L-ornithine pH 7.6, 25°C, mutant enzyme S257A, cofactor: NADPH Aspergillus fumigatus
1.14.13.196 0.14
-
 L-ornithine pH 7.6, 25°C, wild-type enzyme, cofactor: NADPH Aspergillus fumigatus
1.14.13.196 14
-
 NADPH pH 7.6, 25°C, wild-type enzyme Aspergillus fumigatus
1.14.13.196 40
-
 NADPH pH 7.6, 25°C, mutant enzyme S257A Aspergillus fumigatus