Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Biochemistry
Kai TittmannGerhard Hübner

Abstract

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP...Continue Reading

References

Feb 1, 1996·Journal of Molecular Graphics·W HumphreyK Schulten
Jul 10, 1998·Biochimica Et Biophysica Acta·D ChipmanJ V Schloss

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Citations

Nov 8, 2008·Proceedings of the National Academy of Sciences of the United States of America·Piotr NeumannKai Tittmann
Jan 13, 2006·Proceedings of the National Academy of Sciences of the United States of America·Jennifer A McCourtRonald G Duggleby
Jan 11, 2005·Proceedings of the National Academy of Sciences of the United States of America·Kai TittmannDavid M Chipman
Mar 1, 2016·Angewandte Chemie·Thierry LonhienneLuke W Guddat
Feb 1, 2008·Plant Physiology and Biochemistry : PPB·Ronald G DugglebyLuke W Guddat
Aug 28, 2007·Archives of Biochemistry and Biophysics·Kyoung-Jae ChoiMoon-Young Yoon
Aug 2, 2005·Current Opinion in Chemical Biology·David M ChipmanKai Tittmann
Aug 2, 2014·Applied Microbiology and Biotechnology·Jingnan LuAnthony J Sinskey
Sep 1, 2016·Applied Microbiology and Biotechnology·Yadi LiuXiaoyuan Wang
Feb 15, 2018·Proceedings of the National Academy of Sciences of the United States of America·Thierry LonhienneLuke W Guddat
Dec 5, 2019·Chemistry : a European Journal·Oldamur Hollóczki
Feb 6, 2017·The Journal of Biological Chemistry·Thierry LonhienneLuke W Guddat
May 22, 2008·Chemical Reviews·Ronald Kluger, Kai Tittmann
Nov 1, 2005·EcoSal Plus·David P Clark, John E Cronan
Feb 16, 2005·Biochemistry·Jennifer A McCourtRonald G Duggleby

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