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

维生素B12，钴胺素，只是一个家族的成员，这个家族有二十多种不同的相关分子，这些分子被称为钴胺，这些分子只由某些原核生物产生。将钴胺素（B12）与其他变体区别开来并使其成为维生素的原因是在钴胺的较低核苷酸环中存在一种不寻常的碱基，称为5,6-二甲基苯并咪唑（DMB）。这个项目的重点是如何在厌氧条件下产生这种奇怪的碱基（DMB）。维生素是细胞必需的微量营养素，从甲基化和复杂的重排反应到光感应，细胞需要维生素来执行各种生物功能。维生素B12是一种含钴化合物，由连接在较低核苷酸环上的corrin环组成。这种营养物质在维生素中是独特的，因为它只由某些细菌产生。这种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素和甲基钴胺素，它们分别参与重排反应和作为甲基转移酶的辅助因子。这种营养物质实际上属于一个由大约20个相关分子组成的家族，这些分子在钴的下配体性质上都有所不同，我们通常在钴中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组在内的混合细菌群落的营养可利用性和获取中起着重要作用。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）作为其下配体的钴胺素形式，而不是其他20种变体？在这个应用程序中，我们希望解决碱基的合成，DMB，发现在较低的核苷酸环。负责厌氧生物合成的基因已被确定，但该途径的特征仍然很差。令人惊讶的是，对基因簇的生物信息学分析已经确定了两种维生素b12依赖的自由基SAM酶。b12依赖性rSAM酶代表了一个未被充分研究的、催化多样性和极其重要的蛋白质家族。它们是rSAM超家族中最大的酶群之一，并且在许多天然产物（从细菌叶绿素到抗生素和抗癌剂）的生成途径中被发现。此外，在DMB的生物合成中存在依赖B12的酶，这表明维生素参与了自身的合成——换句话说，B12是制造B12所必需的。在这个工作计划中，一系列的实验将为解决这一问题提供机会，这样做将为这些酶如何能够减轻看似不可能的反应提供机制上的见解。该课程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，我们的目标是利用这些获得的知识，并应用合成生物学方法来开发维生素的新变体。该研究将采用最近开发的合成辅因子，并将生产低碱的钴胺素类似物，允许与荧光分子或报告基团下游结合。这将产生一个生化探针工具箱，将用于提高我们对钴胺素贩运的理解，如何获得关键营养素可以调节细菌群落，并提供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.