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，与响应环境中底物消耗的途径方向相比，途径组装向后。所以如果一种酶，E1，催化A到B的反应，那么A就被耗尽了这意味着有能力催化反应产生A的生物体，使用酶E2，从另一种底物中产生A将是有利的。由于E1已经可以结合A，那么E1而不是与A没有亲和力的酶更有可能被复制并突变成E2。本提案的目的是研究主要生化途径/维生素B12（钴胺素）途径中的逆行进化。有人认为，维生素B12（钴胺素）是一种古老的辅酶，在最早的生命形式中使用，在从更古老的RNA世界进化到基于DNA的生命过程中，作为RNA辅助因子发挥着至关重要的作用。可以肯定的是，钴胺素对今天的人类生活至关重要，但令人惊讶的是，我们已经失去了制造钴胺素的能力，而依靠细菌为我们生产钴胺素。细菌中钴胺素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，也因为有趣的化学反应。最典型的例子是环收缩，这是一个独特的过程，其中一个环碳首先从环中挤出，然后完全去除。在corrin环上添加甲基对于指导这种化学反应很重要；负责添加这些基团的酶具有惊人的特异性，可以区分非常相似的底物。这些甲基转移酶序列的相似性表明，7种甲基转移酶中至少有6种是从一个共同的祖先酶进化而来的，该酶被认为催化了所有8种甲基化；我们想了解现代酶如何区分非常相似的底物并执行非常特定的甲基化。这种独特的环收缩过程是由一个甲基的加入引发的，我们想了解这种酶是如何帮助从碳大环中去除一个碳的（催化这一步骤的酶被称为CobJ）。随后，第二个甲基转移酶（称为CobF）在添加甲基的过程中去除挤出的碳。在钴胺素生物合成的30个步骤中，不少于6个是甲基的添加。我们想了解甲基的加入是如何协调钴胺素生物合成的化学过程的。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，并拒绝那些不含钴离子的底物，我们想了解这些酶是如何区分这些底物的。在这项研究结束时，我们将更全面地了解甲基转移酶是如何进化出它们的个体特异性的，以及它们是如何指导产生必需的辅助因子维生素B12的化学反应的。

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.