科研之友
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摘要
Flavin-containing monooxygenases (FMOs) catalyze the oxygenation of diverse organic molecules using OD NADPH, and the flavin adenine dinucleotide (FAD) cofactor. The fungal FMO SidA initiates peptidic siderophore biosynthesis via the highly selective hydroxylation of L-ornithine, while the related amino acid L-lysine is a potent effector of reaction uncoupling to generate H2O2. We hypothesized that protonation states could critically influence both substrate-selective hydroxylation and H2O2 release, and therefore undertook a study of SidA%26apos;s pH-dependent reaction kinetics. Consistent with other FMOs that stabilize a C4a-OO(H) intermediate, SidA%26apos;s reductive half reaction is pH independent. The rate constant for the formation of the reactive C4a-OO(H) intermediate from reduced SidA and O-2 is likewise independent of pH. However, the rate constants for C4a-OO(H) reactions, either to eliminate H2O2 or to hydroxylate L-Orn, were strongly pH-dependent and influenced by the nature of the bound amino acid. Solvent kinetic isotope effects of 6.6 +/- 0.3 and 1.9 +/- 0.2 were measured for the C4a-OOH/H2O2 conversion in the presence and absence of L-Lys, respectively. A model is proposed in which L-Lys accelerates H2O2 release via an acid-base mechanism and where side-chain position determines whether H2O2 or the hydroxylation product is observed.
- 出版日期2014-4-1
