menaquinone-6 and prenol

Prenylated quinones, especially menaquinones, have significant physiological activities, but are arduous to synthesize efficiently. Due to the relaxed aromatic substrate specificity and prenylation regiospecificity at the ortho- site of the phenolic hydroxyl group, the aromatic prenyltransferase NovQ from Streptomyces may be useful in menaquinone synthesis from menadione. In this study, NovQ was overexpressed in Pichia pastoris. After fermentation optimization, NovQ production increased by 1617%. Then the different effects of metal ions, detergents and pH on the activity of purified NovQ were investigated to optimize the prenylation reaction. Finally, purified NovQ and cells containing NovQ were used for menadione prenylation in vitro and in vivo, respectively. Menaquinone-1 (MK-1) was detected as the only product in vitro with γ,γ-dimethylallyl pyrophosphate and menadione hydroquinol substrates. MK-3 at a concentration of 90.53 mg/L was detected as the major product of whole cell catalysis with 3-methyl-2-buten-1-ol and menadione hydroquinol substrates. This study realized whole cell catalysis converting menadione to menaquinones.

Topics: Bacterial Proteins; Biotransformation; Catalysis; Dimethylallyltranstransferase; Hemiterpenes; Hydrogen-Ion Concentration; Metabolic Engineering; Microorganisms, Genetically-Modified; Organophosphorus Compounds; Pentanols; Pichia; Prenylation; Recombinant Proteins; Streptomyces; Substrate Specificity; Vitamin K 2; Vitamin K 3

The analysis of lipid molecules in living organism is an important step in deciphering metabolic pathways. Recently, the zebrafish has been adopted as a valuable animal model system to perform in vivo metabolomics studies, however limited methodologies and protocols are currently available to investigate zebrafish lipidome and even fewer to analyze specific classes of lipids. Here we present an HPLC-HRMS based method to rapidly measure multiple prenol lipid molecules from zebrafish tissues. In particular, we have optimized our method for concurrent detection of ubiquinones (Coenzyme Q6, Coenzyme Q9, Coenzyme Q10), cholesterol, vitamin E (α-tocopherol), vitamin K1 and vitamin K2. The purpose of this study was to compare different ionization modes, mobile phases and stationary phases in order to optimize lipid molecules separation. After HPLC-HRMS parameters selection, several extraction conditions from zebrafish embryos were evaluated. We assessed our methodology by quantitation of analytical recovery on zebrafish extracts from wild-type or zebrafish mutants (barolo) affected by impaired biosynthesis of ubiquinones.

Topics: alpha-Tocopherol; Animals; Cholesterol; Chromatography, High Pressure Liquid; Embryo, Nonmammalian; Hemiterpenes; Mass Spectrometry; Mutation; Pentanols; Ubiquinone; Vitamin E; Vitamin K 1; Vitamin K 2; Zebrafish