||Menaquinone is a lipid-soluble molecule that serves important biological functions. It is an essential electron carrier in the respiratory chain of many microbial pathogens such as Mycobacterium tuberculosis. After decades of research, the menaquinone biosynthesis is best known in E. coli to involve products of eight genes and eight intermediates. In the current study, several mistakes have been identified in the generally accepted menaquinone pathway and biochemical investigations have been performed on the pathway enzymes. Firstly, a new intermediate, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), is identified to be the genuine product of MenD that uses isochorismate and 2-ketoglutarate as substrates. MenD is rediscovered to be an efficient enzyme with a nanomolar Michaelis constant, which may help to direct the flux of the common chorismate precursor into the menaquinone biosynthetic pathway in competition with biosynthesis of aromatic amino acids and other aromatic metabolites. Secondly, through chemoenzymatic synthesis and spectroscopic analysis, this newly identified intermediate is determined to be (1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid. Thirdly, MenH, a previously proposed thioesterase catalyzing a later step in menaquinone biosynthesis, is identified to be responsible for transformation of the new intermediate into (1R,6R)-2-succinyl-6-hydroxy-2,4- cyclohexadiene-1-carboxylic acid by screening the known menaquinone biosynthetic enzymes. Fourthly, MenH is characterized as a/β hydrolase fold enzyme possessing an essential Ser-His-Asp triad typical of serine proteases, but catalyzes a pyruvate elimination reaction initiated by α-proton abstraction from a carbon acid. By using a combination of sequence analysis, site-directed mutagenesis, and computer modeling, the active site of MenH is defined: Arg90, Arg124, and Arg168 are mainly responsible for the binding of substrate through formation of salt bridges with the three negatively charged carboxylate ions in SEPHCHC; side chain polar groups of Tyr85 and Trp147 most likely stabilize the oxyanion generated in the catalytic process through hydrogen bonding; and Lys212 serves as a general acid to facilitate elimination of the pyruvate leaving group. Biochemical studies show that the triad serine residue serves a non-nucleophilic role and may directly abstract the α–proton to initiate the reaction. Moreover, 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA) synthase, or MenB from E. coli, is characterized for the first time and confirmed to lack hydrolytic activity towards DHNA-CoA. Importantly, the synthase is found to involve in a reversible tight-binding interaction with DHNA-CoA and exhibit strong product inhibition. Due to the apparent absence of a dedicated thioesterase in menaquinone biosynthesis, cytosolic thioesterases are suggested to hydrolyze DHNA-CoA as a means of regulation of the pathway by releasing MenB from the strong product inhibition. Finally, the first intermediate in menaquinone biosynthesis, isochorismate, is found to be sequestered by the first two enzymes, MenF and MenD, which involves in protein-protein association in vivo. Higher molecular weight species consisting of menaquinone biosynthetic enzymes have also been found in formaldehyde-fixed E. coli cells, which are indicative of a high level organization of the enzymes in the pathway. These results not only correct the mistakes in the biosynthetic pathway, but also shed lights on catalytic mechanisms of the pathway enzymes as well as regulation of the biogenesis of the important electron transporter naphthenoid.