کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1178688 962710 2012 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Dual role of NADP(H) in the reaction of a flavin dependent N-hydroxylating monooxygenase
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Dual role of NADP(H) in the reaction of a flavin dependent N-hydroxylating monooxygenase
چکیده انگلیسی

Aspergillus fumigatus siderophore A (Af SidA) is a flavin-dependent monooxygenase that catalyzes the hydroxylation of ornithine, producing N5-hydroxyornithine. This is the first step in the biosynthesis of hydroxamate-containing siderophores in A. fumigatus. Af SidA is essential for virulence, validating this enzyme as a drug target. Af SidA can accept reducing equivalents from either NADPH or NADH and displays similar kinetic parameters when using either coenzyme. When the enzyme is reduced with NADPH and reacted with molecular oxygen, a C4a-hydroperoxyflavin intermediate is observed. When the enzyme is reduced with NADH, the intermediate is 2-fold less stable. Steady-state kinetic isotope effect values of 3 and 2 were determined for NADPH and NADH, respectively. The difference in the isotope effect values is due to differences in the rate of flavin reduction by these coenzymes. A difference in the binding mode between these coenzymes was observed by monitoring flavin fluorescence. Limited proteolysis studies show that NADP+, and not NAD+, protects Af SidA from proteolysis, suggesting that it induces conformational changes upon binding. Together, these results are consistent with NADPH having a role in flavin reduction and in the modulation of conformational changes, which positions NADP+ to also play a role in stabilization of the C4a-hydroperoxyflavin.

Figure optionsDownload high-quality image (253 K)Download as PowerPoint slideHighlights
► Af SidA utilizes both NADPH and NADH for flavin reduction.
► NADP+ induces conformational changes, not observed with NAD+.
► These conformational changes are involved in stabilization of flavin intermediates.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics - Volume 1824, Issue 6, June 2012, Pages 850–857
نویسندگان
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