Menaquinone Biosynthesis via the Futalosine Pathway

在化学系生命过程化学项目的支持下，德克萨斯农工大学(TAMU)的Tadhg Begley教授正在研究参与菜籽酮生物合成的酶。在人类和其他哺乳动物中，孟喹酮是一种参与凝血和骨形成的基本维生素(维生素K)。维生素K缺乏会导致凝血时间延长和出血。这些研究将扩大我们对自然反应化学体系的了解，为设计和筛选针对几种人类病原体的新抗生素提供所需的基础科学，并提供一套酶，可用于开发通过发酵生产孟喹酮的新策略。更广泛的影响包括指导德克萨斯农工大学草原景观分校的一名助理教授，为TAMU化学开放日举办一系列生物化学演示，培训本科生和研究生进行生物化学研究，以及开发TAMU有机化学入门课程，这是许多生物科学专业的必修课。这项拟议的研究将完成Futalosine依赖的Menuinone途径上其余四种酶(MqnP、MqnM、MqnL、MqnG)的机制表征。这些酶催化分枝酸转化为1，4-二羟基-6-萘甲酸。生物合成的最后四个步骤的重组和机制表征是这项赠款申请的重点。这些研究将完成所有参与呋喃喹酮生物合成途径的酶的功能分配和机制表征。目的1研究这四种酶在大肠杆菌中的体内重组。目的2研究MqnP催化的戊烯基转位为1，4-二羟基-6-萘甲酸的体外重组，并确定MqnP是否是控制连接的戊烯基团长度的关键酶。目的3研究膜结合的异戊烯基萘酸中间体亲电黄素辅因子介导的脱羧化反应机理。总体而言，需要解决的主要问题是MqnP如何控制维生素的戊烯基取代基的长度，亲电黄素如何介导芳基羧酸的脱羧化，以及MqnK如何实现自由基介导的甲基化。这些问题将通过结构、光谱和衬底模拟研究的组合来回答。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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 prenylgroup. 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.