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教授正在研究与甲基萘醌生物合成有关的酶。在人类和其他哺乳动物中，甲基萘醌是一种必需的维生素（维生素K），参与血液凝固和骨骼形成。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界反应化学的理解，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，并提供一套可用于开发通过发酵生产甲基萘醌的新策略的酶。更广泛的影响包括德克萨斯州农工大学Prairie View分校的一位助理教授的指导，为TAMU化学开放日举办一套生物化学演示，对生物化学研究的本科生和研究生进行培训，以及开发TAMU生物科学专业所需的有机化学入门课程。该研究将完成futalosine依赖性甲基萘醌途径中其余四种酶（MqnP, MqnM, MqnL, MqnG）的机制表征。这些酶催化氯酸盐转化为1,4-二羟基-6-萘酸。生物合成最后四个步骤的重组和机理表征是本拨款申请的重点。这些研究将完成通过futalosine依赖途径参与甲基萘醌生物合成的所有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体内的重组。目的2将重点关注MqnP催化的戊烯基转移到1,4-二羟基-6-环烷酸的体外重构，并确定MqnP是否是控制所附戊烯基长度的关键酶。目的3描述了亲电黄素辅助因子介导的膜结合戊基化萘酸中间体脱羧机制的研究。总的来说，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，以及MqnK如何进行自由基介导的甲基化。这些问题将通过结合结构、光谱和衬底模拟研究来回答。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.