Does enzyme coupling explain the enigma of cobalt reduction during vitamin B12 biosynthesis?

维生素B12是抗恶性贫血因子，与许多复杂的重排和甲基化反应有关。它在生物圈中的重要性可以从我们最近的发现中认识到，超过50%的藻类物种需要这种维生素才能生存，并通过与细菌的共生关系获得维生素。维生素B12的反应中心是钴碳键，它是在corrin环的中心螯合钴离子与腺苷基或甲基之间形成的。因此，在维生素B12的生物合成过程中，这种键是如何形成的，引起了人们相当大的兴趣。在这个应用中，我们希望探索这种钴碳键是如何在维生素B12（钴胺素）的生物合成中形成的。钴胺素也是生物系统中最复杂的“小分子”之一，其复杂性反映在其完全从头合成需要大约30种酶这一事实上。独特的共价金属-碳键是由钴(I)科林斯中间体与ATP反应形成的。这项资助申请的目的是了解这种钴(I)是如何形成的，以及它是如何被呈现给转移腺苷基以产生腺苷钴胺素的酶的。我们克隆了一个特定的corrin还原酶基因，并过量生产了负责将钴（II） corrin还原为钴(I)形式的编码酶，并将其称为CobR。我们是世界上第一个这样做的团队。此外，作为我们初步工作的结果，我们还结晶了酶并解决了它的结构。我们现在希望研究这种酶是如何工作的，并解决CobR如何能够介导一个似乎在能量上不利的反应。为了解决这个问题，我们首先计划研究酶的酶学。我们知道它是一种黄素蛋白，它应该介导一个电子还原。因此，应该有可能检测酶结合的单电子形式（黄酮半醌）的光谱，我们已经概述了一些实验，以帮助我们阐明酶的催化循环。我们还希望了解底物是如何与酶结合的，并找出酶是否通过调节底物结合的方式来调节其催化能力。我们已经概述了一些光谱技术，这些技术将使我们能够直接回答这个问题。最后，我们的初步证据表明，CobR与途径中的下一个酶，腺苷转移酶或CobA相互作用。我们希望获得更多关于这种相互作用的信息，并概述了一系列技术，这些技术将使我们能够在分子细节上研究这两种蛋白质是如何结合的。

Vitamin B12 is the antipernicious anaemia factor that is associated with a number of complex rearrangement and methylation reactions. Its importance in the biosphere can be recognised by our recent finding that more than 50% of all algae species require this vitamin to survive, and acquire the vitamin through a symbiotic relationship with bacteria. The reaction centre of vitamin B12 is the cobalt-carbon bond, which is formed between the centrally chelated cobalt ion of the corrin ring and either an adenosyl group or a methyl group. There is thus some considerable interest in how this bond is made during the biosynthesis of vitamin B12. In this application we wish to explore how this cobalt-carbon bond is formed in the biosynthesis of vitamin B12 (cobalamin). Cobalamin is also one of the most complex 'small molecules' made in biological systems, a complexity that is reflected in the fact that about thirty enzymes are required for its complete de novo synthesis. The unique covalent metal-carbon bond is formed by reacting a cobalt (I) corrin intermediate with ATP. The aim of this grant application is to understand how this cobalt (I) form is made and how it is then presented to the enzyme that transfers the adenosyl group to generate adenosylcobalamin. We have cloned a specific corrin reductase gene and overproduced the encoded enzyme that is responsible for the reduction of the cobalt (II) corrin species to the cobalt (I) form, and have called it CobR. We are the first group in the world to have done this. Moreover, as a result of our preliminary work, we have also crystallised the enzyme and solved its structure. We now wish to investigate how the enzyme works and address how CobR is able to mediate a reaction that appears to be energetically unfavourable. To address this problem, we first plan to investigate the enzymology of the enzyme. We know that it is a flavoprotein and that it should mediate a one electron reduction. Thus it should be possible to detect the enzyme-bound one electron form (a flavosemiquinone) spectroscopically and we have outlined a number of experiments to help us elucidate the catalytic cycle of the enzyme. We also wish to understand how the substrate binds to the enzyme, and to find out if the enzyme mediates it catalytic power by modulating the way in which the substrate binds. We have outlined a number of spectroscopic techniques that will allow us to answer this directly. Finally, our preliminary evidence suggests that CobR interacts with the next enzyme in the pathway, the adenosyltransferase or CobA. We wish to obtain more information on this interaction and have outlined a range of techniques that will allow us to investigate in molecular detail how the two proteins may associate.