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.

A healthy lifestyle may ameliorate metabolic syndrome (MetS); however, it remains unclear if incorporating nuts or seeds into lifestyle counseling (LC) has additional benefit. A 3-arm, randomized, controlled trial was conducted among 283 participants screened for MetS using the updated National Cholesterol Education Program Adult Treatment Panel III criteria for Asian Americans. Participants were assigned to a LC on the AHA guidelines, LC + flaxseed (30 g/d) (LCF), or LC + walnuts (30 g/d) (LCW) group. After the 12-wk intervention, the prevalence of MetS decreased significantly in all groups: -16.9% (LC), -20.2% (LCF), and -16.0% (LCW). The reversion rate of MetS, i.e. those no longer meeting the MetS criteria at 12 wk, was not significantly different among groups (LC group, 21.1%; LCF group, 26.6%; and LCW group, 25.5%). However, the reversion rate of central obesity was higher in the LCF (19.2%; P = 0.008) and LCW (16.0%; P = 0.04) groups than in the LC group (6.3%). Most of the metabolic variables (weight, waist circumference, serum glucose, total cholesterol, LDL cholesterol, apolipoprotein (Apo) B, ApoE, and blood pressure) were significantly reduced from baseline in all 3 groups. However, the severity of MetS, presented as the mean count of MetS components, was significantly reduced in the LCW group compared with the LC group among participants with confirmed MetS at baseline (P = 0.045). Our results suggest that a low-intensity lifestyle education program is effective in MetS management. Flaxseed and walnut supplementation may ameliorate central obesity. Further studies with larger sample sizes and of longer duration are needed to examine the role of these foods in the prevention and management of MetS.

Menaquinone is a lipid-soluble molecule that plays an essential role as an electron carrier in the respiratory chain of many bacteria. We have previously shown that its biosynthesis in Escherichia coli involves a new intermediate, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and requires an additional enzyme to convert this intermediate into (1 R,6 R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC). Here, we report the identification and characterization of MenH (or YfbB), an enzyme previously proposed to catalyze a late step in menaquinone biosynthesis, as the SHCHC synthase. The synthase catalyzes a proton abstraction reaction that results in 2,5-elimination of pyruvate from SEPHCHC and the formation of SHCHC. It is an efficient enzyme ( k cat/ K M = 2.0 x 10 (7) M (-1) s (-1)) that provides a smaller transition-state stabilization than other enzymes catalyzing proton abstraction from carbon acids. Despite its lack of the proposed thioesterase activity, the SHCHC synthase is homologous to the well-characterized C-C bond hydrolase MhpC. The crystallographic structure of the Vibrio cholerae MenH protein closely resembles that of MhpC and contains a Ser-His-Asp triad typical of serine proteases. Interestingly, this triad is conserved in all MenH proteins and is essential for the SHCHC synthase activity. Mutational analysis found that the catalytic efficiency of the E. coli protein is reduced by 1.4 x 10 (3), 2.1 x 10 (5), and 9.3 x 10 (3) folds when alanine replaces serine, histidine, and aspartate of the triad, respectively. These results show that the SHCHC synthase is closely related to alpha/beta hydrolases but catalyzes a reaction mechanistically distinct from all known hydrolase reactions.