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.
SidA (siderophore A) is a flavin-dependent N-hydroxylating monooxygenase that is essential for virulence in Aspergillus fumigatus. SidA catalyzes the NADPH- and oxygen-dependent formation of N(5)-hydroxyornithine. In this reaction, NADPH reduces the flavin, and the resulting NADP(+) is the last product to be released. The presence of NADP(+) is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species. As part of our efforts to determine the molecular details of the role of NADP(H) in catalysis, we targeted Ser-257 for site-directed mutagenesis and performed extensive characterization of the S257A enzyme. Using a combination of steady-state and stopped-flow kinetic experiments, substrate analogs, and primary kinetic isotope effects, we show that the interaction between Ser-257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin. Molecular dynamics simulation results suggest that Ser-257 functions as a pivot point, allowing the nicotinamide of NADP(+) to slide into position for stabilization of the C4a-hydroperoxyflavin.
From the Department of Biochemistry.
Carolyn
C
Shirey
From the Department of Biochemistry.
Somayesadat
S
Badieyan
From the Department of Biochemistry; the Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061. Electronic address: psobrado@vt.edu.
Pablo
P
Sobrado
D019943:Amino Acid Substitution; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D005415:Flavins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009249:NADP / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C531259:C4a-hydroperoxyflavin; D005415:Flavins; D005656:Fungal Proteins; D012694:Serine; D009249:NADP; D006899:Mixed Function Oxygenases; C059419:ornithine N5-oxygenase
Aspergillus; C4a-hydroperoxyflavin; Flavin; Flavin-dependent Monooxygenase; Molecular Dynamics; Mutagenesis; N-Hydroxylating; NADP(H) Role; SidA; Siderophores