Studies on Vitamin K Biosynthesis

实验（申请人提供）：人类和动物不能合成 维生素K（K）。依赖钾的低凝血原血症是临床上的一个重要问题 在一些条件下。人类和动物营养中的钾需要量 是由绿色植物和肠道细菌满足的。因为K扮演着重要的角色 在人类健康中的作用，重要的是要了解其生物合成和 调控 维生素K的生物合成途径可以总结如下： 分支酸酯-menF->异分支酸酯-menD-> 2-琥珀酰基-6-羟基-2， 4-环己二烯-1-羧酸酯（SHCHC）-- （OSB）-menE->邻-琥珀酰基苯甲酰基-CoA（OSB-CoA）-menB->{ 1， 4-二羟基-2-萘甲酰基-CoA（DHNA-CoA）} 酸（DHNA）-menA->去甲基甲萘醌（DMK）- ubiE -->甲萘醌 （MK）。 7个ORF（其中6个已被证明是甲基萘醌生物合成基因） 按照orf 1 O 1、menF、menD、menH、menB、menC和menE的顺序排列在一个簇中。 所有基因的转录都是由一个强启动子启动的 在厌氧条件下检测到orf 101 5'端的26 bp。menA基因 位于染色体上89分钟处，与一个未知的ORF连锁 （orf161）。 在本研究中，我们提出：1）确定orf 101的作用; 2）研究orf 101的作用机制。 使用lacZ融合物调节men操纵子; 3）确定结构 拟定中间体（DHNA-CoA）; 4）鉴定甲基转移酶 参与DMK->MK转化，以及5）表征活性位点 OSB合酶的突变体，其三维结构最近被解决 被我们

DESCRIPITON (provided by applicant): Humans and animals cannot synthesize vitamin K (K). K dependent, hypoprothrombia is an important clinical problem under a number of conditions. The K requirement in human and animal nutrition is met by green plants and intestinal bacteria. Because K plays an important role in human well being, it is important to understand its biosynthesis and regulation. The vitamin K biosynthetic pathway may be summarized as follows: chorismate -menF--> isochorismate --menD--> 2-succinyl-6-hydroxy-2, 4-cyclohexadiene-1 -carboxylate (SHCHC) ---menC---->o-succinylbenzoic acid (OSB)-menE-> o-succinylbenzoyl-CoA (OSB-CoA) --menB-->{ 1, 4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA)}---menH---> 1, 4-dihydroxy-2-naphthoic acid (DHNA) ---menA---> demethylmenaquinone (DMK) --- ubiE --> menaquinone (MK). Seven ORFs (six of which have been shown to be menaquinone biosynthetic genes) lie in a cluster in the order orf1O1, menF, menD, menH, menB, menC, and menE. Transcription of all the genes are initiated from a single strong promoter site, 26 bp 5' to orfiOl was detected under anaerobic conditions. The menA gene is located at 89 min on the chromosome and is linked to an unidentified orf (orf161). In this study, we propose 1) to determine the role of orf101; 2) to study the regulation of the men operon using lacZ fusions; 3) to determine the structure of a proposed intermediate (DHNA-CoA); 4) to identify the methyltranferase involved in the DMK-->MK conversion, and 5) to characterize the active site mutants of OSB synthase, whose three dimensional structure was recently solved by us.