A corrin conundrum: Is vitamin B12 required for its own biogenesis?

维生素B12，钴胺，只是被称为钴酰胺的二十多种不同相关分子家族中的一员，这些分子只由某些原核生物独有地制造。钴胺(B12)的不同之处在于钴酰胺的下核苷酸环中存在一个不寻常的碱基，称为5，6-二甲基苯并咪唑(DMB)。这个项目的重点是这个奇怪的碱基(DMB)是如何在厌氧条件下形成的。维生素是细胞执行各种生物学功能所需的基本微量营养素，从甲基化和复杂的重排反应到光感知。维生素B12是一种含钴化合物，由连接在较低核苷酸环上的科林环组成。这种营养素在维生素中是独一无二的，因为它只由某些细菌产生。该合成是通过一个高度复杂的生物合成途径进行的，涉及大约30个酶介导的步骤。维生素的生物活性形式最常见的是腺苷钴胺和甲钴胺，它们分别参与重排反应和作为甲基转移酶的辅助因子。这种营养素实际上属于一个由大约20个相关分子组成的家族，这些分子的低配体与钴的性质都不同，我们通常会发现苯并咪唑衍生物、嘌呤衍生物和苯酚等芳香族化合物。这种多样性在包括人类微生物群在内的混合细菌群落中的营养供应和获取方面发挥着重要作用。一个关键的问题是，为什么真核生物只选择含有5，6-二甲基苯并咪唑(DMB)的钴胺形式作为其下位配体，而不是其他20种变体？在本申请中，我们希望解决在低核苷酸环中发现的碱基DMB的合成。负责厌氧生物合成的基因已经确定，但其途径仍不清楚。令人惊讶的是，对该基因簇的生物信息学分析发现了两种依赖维生素B12的自由基SAM酶。依赖于B12的rSAM酶代表了一个未被充分研究、催化多样性和极其重要的蛋白质家族。它们形成了rSAM超家族中最大的一组酶，并在从细菌叶绿素到抗生素和抗癌剂的许多天然产物的途径中都被鉴定出来。此外，在DMB的生物合成中存在依赖于B12的酶，这表明维生素参与了自身的合成--换句话说，B12是合成B12所必需的。在这项工作计划中，概述了一系列实验，这些实验将提供一个解决这一问题的机会，并通过这样做将提供对这些酶如何能够缓解似乎不可能的反应的机械性见解。该计划的前三个实验部分涉及该途径的生物化学和酶学。在最后一节中，我们的目标是利用所获得的知识，并应用合成生物学方法来开发维生素的新变种。这项研究将使用最近开发的合成辅因子，并将生产更低碱基的钴胺类似物，允许下游与荧光分子或报告基团连接。这将产生一个生化探针工具箱，用于提高我们对钴胺贩运的理解，了解获得关键营养素如何调节细菌群落，并提供有关维生素在疾病过程中的作用的信息。

Vitamin B12, cobalamin, is just one member of a family of over twenty different related molecules that are called cobamides, molecules that are exclusively made by only certain prokaryotes. What differentiates cobalamin (B12) and makes it a vitamin from these other variants is the presence of an unusual base in the lower nucleotide loop of the cobamide called 5,6-dimethylbenzimidazole (DMB) This project is focussed on how this curious base (DMB) is made under anaerobic conditions. Vitamins are essential micronutrients that are required by cells to perform a diverse range of biological functions, from methylation and complex rearrangement reactions through to light sensing. Vitamin B12 is a cobalt-containing compound that is composed of a corrin ring attached to a lower nucleotide loop. The nutrient is unique among the vitamins in that it is made exclusively by only certain bacteria. The synthesis is orchestrated via a highly complex biosynthetic pathway involving around thirty enzyme-mediated steps. The biologically active forms of the vitamin are most commonly adenosylcobalamin and methylcobalamin, which are involved in rearrangement reactions and as a cofactor for methyltransferases respectively. The nutrient actually belongs to a family of around 20 related molecules that all differ in the nature of the lower ligand to the cobalt, where we typically find benzimidazole derivatives, purine derivatives and aromatics such as phenol. This diversity plays an important role in nutrient availability and acquisition in mixed bacterial communities which include the human microbiome. A key question is why eukaryotes have exclusively selected the form of the cobalamin which contains 5,6-dimethylbenzimidazole (DMB) as its lower ligand over the other twenty variants? In this application we wish to address the synthesis of the base, DMB, found in the lower nucleotide loop. The genes responsible for the anaerobic biosynthesis have been identified but the pathway remains poorly characterised. Surprisingly, bioinformatic analysis of the gene cluster has identified two vitamin B12-dependent radical SAM enzymes. B12-dependent rSAM enzymes represent an understudied, catalytically diverse and incredibly important family of proteins. They form one of the largest groups of enzymes within the rSAM superfamily and have been identified in the pathways of many natural products from bacteriochlorophyll to antibiotics and anticancer agents. Moreover, the presence of B12-dependent enzymes in the biosynthesis of DMB suggests that the vitamin is involved in its own synthesis - in other words B12 is required to make B12. In this program of work, a series of experiments are outlined that will provide an opportunity to address this point and in so doing will provide mechanistic insights into how these enzymes are able to mitigate seemingly impossible reactions. The first three experimental sections of the programme deal with the biochemistry and enzymology of the pathway. In the final section we aim to use this gained knowledge, and apply synthetic biology approaches to develop novel variants of the vitamin. The research will employ recently developed synthetic cofactors and will produce lower base analogues of cobalamin which allow for downstream conjugation with fluorescent molecules or reporter groups. This will generate a tool box of biochemical probes which will be used to improve our understanding of the trafficking of cobalamin, how access to key nutrients can regulate bacterial communities and also provide information on the role of the vitamin in disease processes.