Elucidation and evolution of substrate recognition and reaction mechanism in the methyltransferases of cobalamin biosynthesis

1945年，霍洛维茨提出了最早描述代谢途径进化的理论之一--逆行进化模型。它指出，在进化过程中，与路径方向相比，路径向后组装，以响应环境中底物的耗尽。因此，如果一种酶E1催化A到B的反应，那么A就会耗尽，这意味着有能力催化反应从另一种底物产生A的有机体将处于优势。由于E1已经可以与A结合，那么更有可能的是，E1而不是一种与A没有亲和力的酶会被复制并突变成E2。这项建议的目的是研究主要生化途径/维生素B12(钴胺)途径的逆行进化。有人认为维生素B12(钴胺)是一种古老的辅酶，用于最早的生命形式，在更古老的RNA世界中作为RNA辅助因子在基于DNA的生命进化中发挥着重要作用。可以肯定的是，钴胺对今天的人类生活是必不可少的，那么也许令人惊讶的是，我们已经失去了制造钴胺的能力，而是依赖细菌为我们制造钴胺。钴胺在细菌中的生物合成是令人着迷的，因为它的复杂性，生物合成涉及大约30种酶，也因为涉及的有趣的化学物质。最好的例子是环收缩，这是一种独特的过程，在这个过程中，其中一个环碳首先从环上挤出，然后完全去除。在Corin环上添加甲基对指导这一化学过程很重要；负责添加这些基团的酶是令人惊讶的特定的，在非常相似的底物之间区分开来。这些甲基转移酶序列的相似性表明，七种甲基转移酶中至少有六种是从一种共同的祖先酶进化而来的，这种酶被认为催化了所有八种甲基化；我们想要了解现代酶是如何区分非常相似的底物并执行非常特定的甲基化的。独特的环收缩过程是由添加一个甲基触发的，我们想了解酶是如何帮助从碳大循环中移除一个碳的(催化这一步骤的酶被称为CobJ)。第二个甲基转移酶(称为CobF)随后在一个过程中移除挤出的碳，这个过程再次由甲基的添加触发。钴胺生物合成的30个步骤中至少有6个是甲基的加成。我们想了解甲基的加入是如何协调钴胺生物合成的化学的。我们的研究表明，一些酶可以选择含有钴离子的四吡咯底物，而拒绝那些不含钴离子的底物，我们想了解酶是如何区分这些底物的。在这项研究的最后，我们将对甲基转移酶如何进化它们的个体特异性以及它们如何指导导致基本辅因子维生素B12的化学过程有一个更完整的了解。

In 1945, Horowitz proposed one of the first theories describing the evolution of metabolic pathways, the retrograde evolution model. It states that during evolution pathways assembled backwards compared to the pathway direction in response to depletion of substrates in the environment. So if an enzyme, E1, catalyses the reaction A to B, then A is depleted which means an organism with the ability to catalyse a reaction producing A, using enzyme E2, from another substrate would be at an advantage. Since E1 can already bind A then there is a greater chance that E1 rather an enzyme without affinity to A would be duplicated and mutated into E2. The aim of this proposal is to investigate retrograde evolution within a major biochemical pathway / the Vitamin B12 (cobalamin) pathway. It has been argued that Vitamin B12 (cobalamin) is an ancient coenzyme that was used in the earliest forms of life playing an essential role as an RNA cofactor in the evolution of DNA based life from the more ancient RNA world. What is certain is that cobalamin is essential for human life today, it is perhaps surprising then that we have lost the ability to make cobalamin and rely on bacteria to produce it for us. The biosynthesis of cobalamin in bacteria is fascinating because of its complexity, the biosynthesis involves some 30 enzymes, and also because of the interesting chemistry involved. The prime example is ring contraction, a unique process in which one of the ring carbons is first extruded from the ring and later removed completely. The addition of methyl groups to the corrin ring is important in directing this chemistry; the enzymes responsible for adding these groups are surprisingly specific, distinguishing between closely similar substrates. Similarities in the sequences of these methyltransferases shows that no fewer than six of the seven methyltransferase enzymes have evolved from a common ancestral enzyme which is presumed to have catalysed all eight methylations; we want to understand how the modern enzymes discriminate between closely similar substrates and perform very specific methylations. The unique ring contraction process is triggered by the addition of one of the methyl groups and we want to understand how the enzyme helps to remove one carbon from the carbon macrocycle (the enzyme that catalyses this step is called CobJ). A second methyltransferase (called CobF) subsequently removes the extruded carbon in a process again triggered by the addition of a methyl group. No fewer than six of the 30 steps in cobalamin biosynthesis are the addition of methyl groups. We want to understand how the addition of the methyl groups orchestrates the chemistry of cobalamin biosynthesis. Our studies show that some of the enzymes can select tetrapyrrole substrates containing a colbalt-ion and reject those that don't and we want to understand how the enzymes discriminate between these substrates. At the end of this research we will have a more complete understanding of how the methyltransferases evolved their individual specificities and the how they direct the chemistry that results in the essential cofactor vitamin B12.