Award details

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

Reference	BB/E002137/1
Principal Investigator / Supervisor	Professor Richard Pickersgill
Co-Investigators / Co-Supervisors	Dr Stephen Rigby, Professor Martin Warren
Institution	Queen Mary University of London
Department	Sch of Biological and Chemical Sciences
Funding type	Research
Value (£)	375,077
Status	Completed
Type	Research Grant
Start date	01/05/2007
End date	30/04/2010
Duration	36 months

Abstract

The biosynthesis of cobalamin is remarkable because of the complexity of the biosynthetic pathway, the chemistry involved, and because it may have evolved via retrograde evolution. The addition of 8 methyl groups to the ring carbons of the corrin macrocycle accounts for no fewer than 6 of the 30 enzyme-mediated steps in the biosynthesis of cobalamin from 5-aminolevulinic acid. Within the aerobic pathway six of the seven methyltransferases have sequence similarity clearly indicating that these enzymes have evolved from a common ancestor. Indeed, most of the methyltransferases on the aerobic route have an equivalent in the anaerobic pathway, indicating a split after the primordial vitamin B12 pathway had evolved. The canonical methyltransferases may be a splendid example of retrograde evolution. The addition of methyl groups prevents reversible C-protonation and leads to the formation of particular tautomeric intermediates. The specific spatial and temporal addition of eight methyl groups on the macrocycle reveals the importance of forming the correct double bond configurations essential for much of the chemistry that is required for corrin ring synthesis. The coupling of methylation at C-17 and ring contraction (CobJ) and methylation at C-1 and deacylation (CobF) are particularly interesting exemplars. It has been suggested that methylation at C-17 in the substrate precorrin 3b results in the ketone function pendant from C-1 by a pinacol rearrangement. We shall investigate this possibility and establish the role the enzyme takes in promoting the rearrangement and investigate the role of CobF in promoting deacylation. The discrimination between similar substrates is remarkable, apparently dependent on the order of the proportionate and acetate side chains and presence or absence of a metal-ion within the macrocycle. We plan to understand the molecular basis of the discrimination between these substrates.

Summary

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.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search