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

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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.