title: Menaquinone biosynthesis in Escherichia coli: identification of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate as a novel intermediate and re-evaluation of MenD activity.
abstract: Menaquinone is an electron carrier in the respiratory chain of Escherichia coli during anaerobic growth. Its biosynthesis involves (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) as an intermediate, which is believed to be derived from isochorismate and 2-ketoglutarate by one of the biosynthetic enzymes-MenD. However, we found that the genuine MenD product is 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid (SEPHCHC), rather than SHCHC. This is supported by the following findings: (i) isochorismate consumption and SHCHC formation are not synchronized in the enzymic reaction, (ii) the rate of SHCHC formation is independent of the enzyme concentration, (iii) SHCHC is not formed in weakly acidic or neutral solutions in which the isochorismate substrate is readily consumed by MenD, and (iv) the MenD turnover product, formed under conditions disabling SHCHC formation, possesses spectroscopic characteristics consistent with the structure of SEPHCHC and spontaneously undergoes 2,5-elimination to form SHCHC and pyruvate in weakly basic solutions. Two properties of the intermediate, ultraviolet transparency and chemical instability, provide a rationale for the fact that SHCHC has been consistently mistaken as the MenD product. In accordance with these findings, MenD was rediscovered to be a highly efficient enzyme with a high second-order rate constant and should be renamed SEPHCHC synthase. Intriguingly, the enzymatic activity responsible for conversion of SEPHCHC into SHCHC appears not to associate with any of the known enzymes in menaquinone biosynthesis but is present in the crude extract of E. coli K12, suggesting that a genuine SHCHC synthase remains to be identified to fully elucidate the ubiquitous biosynthetic pathway.
authors.affiliation: Department of Chemistry, Center for Cancer Research, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Jiang
authors.forename: Yang
authors.initials: Y
authors.lastname: Cao
authors.forename: Zu-Feng
authors.initials: ZF
authors.lastname: Guo
authors.forename: Minjiao
authors.initials: M
authors.lastname: Chen
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Chen
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Guo
mesh_terms: D053898:Biosynthetic Pathways / Q000502:physiology*; D002384:Catalysis; D002827:Chorismic Acid / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003509:Cyclohexanecarboxylic Acids / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003510:Cyclohexanes; D053138:Cyclohexenes / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007651:Keto Acids / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D012015:Reference Standards; D012459:Salicylates / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013386:Succinates / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D053719:Tandem Mass Spectrometry; D009626:Terminology as Topic; D024482:Vitamin K 2 / Q000737:chemistry / Q000378:metabolism*
chemical_list: C524466:2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid; C087010:2-succinyl-6-hydroxycyclohexa-2,4-diene-1-carboxylic acid; D003509:Cyclohexanecarboxylic Acids; D003510:Cyclohexanes; D053138:Cyclohexenes; D029968:Escherichia coli Proteins; D007651:Keto Acids; D012459:Salicylates; D013386:Succinates; D024482:Vitamin K 2; C052985:isochorismic acid; C060510:2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase; D007652:Oxo-Acid-Lyases; D002827:Chorismic Acid