Menaquinone Biosynthesis via the Futalosine Pathway

Texas A&M University, College Station TX

Investigators

Abstract

With the support of the Chemistry of Life Processes program in the Division of Chemistry, Professor Tadhg Begley from Texas A&M University (TAMU) is studying the enzymes involves in the biosynthesis of menaquinone. In humans and other mammals, menaquinone is an essential vitamin (vitamin K) involved in blood clotting and bone formation. Vitamin K deficiency leads to prolonged clotting time and hemorrhaging. These studies will broaden our understanding of nature's repertoire of reaction chemistry, provide the basic science needed for the design and screening of new antibiotics against several human pathogens and make available a set of enzymes that can be used for the development of new strategies to produce menaquinone by fermentation. The broader impacts include the mentoring of an assistant professor at Texas A&M University Prairie View, the hosting of a set of biological chemistry demos for the TAMU Chemistry open house, the training of undergraduate and graduate students in biological chemistry research and the development of the TAMU introductory organic chemistry course required for numerous biological science majors. The proposed research will complete the mechanistic characterization of the remaining four enzymes on the futalosine-dependent menaquinone pathway (MqnP, MqnM, MqnL, MqnG). These enzymes catalyze the conversion of chorismate to 1,4-dihydroxy-6-naphthoic acid. The reconstitution and mechanistic characterization of the last four steps of the biosynthesis is the focus of this grant application. These studies will complete the functional assignment and mechanistic characterization of all the enzymes involved in the biosynthesis of menaquinone by the futalosine-dependent pathway. Aim 1 will study the reconstitution of the four enzymes in vivo in Escherichia coli. Aim 2 will focus on the in vitro reconstitution of the MqnP-catalyzed prenyl transfer to 1,4-dihydroxy-6-naphthoic acid and determine if MqnP is the key enzyme controlling the length of the attached prenyl group. Aim 3 describes studies on the mechanism of the electrophilic-flavin-cofactor-mediated decarboxylation of the membrane bound prenylated naphthoic acid intermediate. Overall, the major questions to be addressed are how MqnP controls the length of the vitamin’s prenyl substituent, how electrophilic-flavin mediates the decarboxylation of aryl carboxylic acids, and how MqnK carries out a radical-mediated methylation of menaquinone. These questions will be answered using a combination of structural, spectroscopic and substrate analogue studies. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.