Biosynthesis of Menaquinone in E. coli: Identification of an Elusive Isomer of SEPHCHC

Abstract

In the biosynthesis of menaquinone in bacteria, the thiamine diphosphate-dependent enzyme MenD catalyzes the decarboxylative carboligation of α-ketoglutarate and isochorismate to (1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC). The regioisomer of SEPHCHC, namely (1R,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-2-ene-1-carboxylate (iso-SEPHCHC), has been considered as a possible product, however, its existence has been doubtful due to a spontaneous elimination of pyruvate from SEPHCHC to 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC). In this work, the regioisomer iso-SEPHCHC was distinguished from SEPHCHC by liquid chromatography-tandem mass spectrometry. Iso-SEPHCHC was purified and identified by NMR spectroscopy. Just as SEPHCHC remained hidden as a MenD product for more than two decades, its regioisomer iso-SEPHCHC has remained until now.