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

Corrin 难题：维生素 B12 自身的生物发生是否需要维生素 B12？

• 批准号: BB/V002252/2
• 负责人: Andrew Lawrence
• 金额: $ 32.15万
• 依托单位: University of Southampton
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV002252%2F2
• 关键词: corrin; conundrum; vitamin; B12; required

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.

维生素B12，钴胺素，只是一个家庭的成员，超过20种不同的相关分子，被称为钴酰胺，分子是专门由某些原核生物。钴胺素（B12）的区别并使其成为维生素与其他变体的区别是在钴酰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）该项目的重点是如何在厌氧条件下制造这种奇怪的碱基（DMB）。维生素是细胞执行各种生物功能所需的必需微量营养素，从甲基化和复杂的重排反应到光传感。维生素B12是一种含钴化合物，由连接到较低核苷酸环的咕啉环组成。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制成。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因子。这种营养素实际上属于大约20种相关分子的家族，这些分子在钴的低级配体的性质上都不同，我们通常会发现苯并咪唑衍生物，嘌呤衍生物和芳香族化合物，如苯酚。这种多样性在包括人类微生物组在内的混合细菌群落中的营养物质可用性和获取方面起着重要作用。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）作为其较低配体的钴胺素形式，而不是其他20种变体？在本申请中，我们希望解决在较低的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被确定，但该途径仍然缺乏特征。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依赖性自由基SAM酶。B12依赖性rSAM酶代表了一个研究不足，催化多样性和令人难以置信的重要蛋白质家族。它们形成rSAM超家族中最大的酶组之一，并且已经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被鉴定。此外，在DMB的生物合成中存在B12依赖性酶，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。在本工作计划中，概述了一系列实验，这些实验将提供解决这一点的机会，从而提供有关这些酶如何能够减轻看似不可能的反应的机制见解。该计划的前三个实验部分涉及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，并将产生钴胺素的低碱基类似物，其允许与荧光分子或报告基团的下游缀合。这将产生一个生化探针工具箱，用于提高我们对钴胺素贩运的理解，如何获得关键营养素来调节细菌群落，并提供有关维生素在疾病过程中的作用的信息。