The reversible dehydration of (R)-2-hydroxyglutarate to (E)-glutaconate

Abstract

1. During fermentation with whole cells of Acidaminococcus fermentans or Clostridium microsporum the pro-3S hydrogen of (R)-2-hydroxyglutarate or of its precursor (S)-glutamate is eliminated stereospecifically. Since (E)-glutaconate but not its Z isomer is fermented by whole cells or cell-free extracts of A. fermentans, the overall dehydration of (R)-2-hydroxyglutarate to (E)-glutaconate can be described as syn. 2. The fermentation of (E)-glutaconate required acetyl phophate, CoA and NAD, that of (S)-glutamate or (R)-2-hydroxyglutarate additionally MgCl2, FeSO4 and dithioerythritol. The fermentations of all three substrates were inhibited by avidin and stimulated by biotin. 3. The hydration of (E)-glutaconate was measured enzymically by the formation of (R)-2-hydroxyglutarate. The dehydration of the hydroxy acid was assayed by the release of 3HOH from (2R)-2-hydroxy[3-3H]glutarate. Optimum conditions were found by activation of the cell-free extract with MgCl2, FeSO4, dithioerythritol, acetyl phosphate anmd NADH followed by the reaction which only required acetyl phosphate and CoA as cofactors. Activation and reaction had to be performed anaerobically. 4. The dehydration was inhibited by 2 mM azide, 1 mM arsenate, 1 mM hydroxylamine, 20 micro M dinitrophenol or 10 micro M carbonylcyanide p-trifluoromethoxyphenylhydrazone. 5. It is concluded that the actual substrates of the dehydration are the corresponding thiol esters. The data indicate a catalytical phosphorylation during the reaction.