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Flavin dehydration in a class B monooxygenase
Author(s) -
Sobrado Pablo,
Robinson Reeder,
Britt Korliss
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.00166
Subject(s) - flavin group , chemistry , monooxygenase , stereochemistry , enzyme , turnover number , steady state (chemistry) , biochemistry , active site , organic chemistry , cytochrome p450
Siderophore A (SidA) is an ornithine hydroxylase that belongs to the class B flavin monooxygenase family of enzymes. SidA catalyzes the first step in the biosynthesis of hydroxamate‐containing siderophores in Aspergillus fumigatus . Previous structural analysis revealed that R144, a residue adjacent to the FAD, undergoes redox‐dependent conformational changes. In order to determine the role of R144 in catalysis, site‐directed mutagenesis was used to create the R144A enzyme. Steady‐state kinetic measurements showed that the mutant variant R144A has a 20‐fold lower turnover number. Pre‐steady‐state kinetic measurements, pH profiles, and solvent kinetic isotope effect measurements were used to isolate the microscopic step responsible for the reduced steady‐state activity. The data are consistent with flavin dehydration being the main step affected by the mutation. We proposed that R144 plays a role, at least in part, in providing the proper hydrogen bonding network for the elimination of the hydroxide from the flavin‐hydroxide intermediate. Implication for a conserved mechanism in other flavin‐dependent enzymes is discussed.