Mechanistic investigations into the novel monooxygenases CobG and CobZ involved in vitamin B12 biosynthesis

Vitamins are essential nutrients required by humans to complete their diet, and by definition are not made within their own body. One of the most interesting aspects of vitamin B12 metabolism is that this essential nutrient is made only by bacteria / no higher order organism (eukaryote) has the ability to make this compound. Whilst some eukaryotes have retained a role for vitamin B12 in their metabolism, others such as plants have evolved into a life without B12. A consequence of this is that those on strictly vegetarian diets are prone to vitamin B12 deficiency - a state that is associated with a wide range of symptoms including megablastic anaemia, neurological disorders, and developmental problems in unborn babies. Vitamin B12 deficiency is also a problem in the elderly, where an increase in the level of B12 in the diet can alleviate the symptoms. There are thus strong medical reasons for understanding how this vitamin is made so that we can devise ways to make more and thus increase the levels of vitamin B12 in the diet. In this application we wish to explore one important stage in the biosynthesis of vitamin B12. Vitamin B12 is made by a factory of enzymes / in fact it takes about thirty different enzymes to make this essential compound. Enzymes are biological catalysts that speed up reactions that go on inside cells / without enzymes many reactions would take over a decade to occur instead of just a few seconds. In common with all enzymes, the enzymes responsible for the biosynthesis of vitamin B12 show specificity. This means that each enzyme in the vitamin B12 biosynthetic pathway only catalyses one reaction / it is specific for that reaction only. This then leads to the question of how such large pathways may have arisen? One current theory is that some enzymes are adaptable / that is they catalyse general reactions and only become highly specific later on. We have identified one step in the biosynthesis of vitamin B12 that seems to have acquired two quite distinct enzymes to catalyse one reaction. We wish to study the action of these two enzymes in order to understand how they have adapted to the vitamin B12 biosynthetic pathway. This application is aimed at increasing our understanding of how biochemical pathways operate, how they are controlled and how they can be engineered to enhance the metabolic ability of the host cell. From medical, industrial and wealth creation strategic standpoints, this research programme closely follows the remit and aspirations of the BBSRC.

This research application is appropriate for the Biomolecular Sciences Committee since the research at the interface between biology and chemistry, involving studies at the molecular level. The theme of the application is biological catalysis, since it aims to study the mechanisms and thermodynamics of enzyme catalysis in the biosynthesis of vitamin B12, including the elucidation of the function and mechanisms of action of metal and coenzyme containing biological enzymes. In particular, the application falls under the priority of Mechanistic Enzymology (MENZ). Within this project we wish to study the mechanism of two enzymes, CobG and CobZ. Both enzymes catalyse the same reaction within the biosynthesis of vitamin B12, and are associated with the chemically daunting process of ring contraction. Specifically, they act as monooxygenases and catalyse the hydroxylation of a specific intermediate. However, the enzymes mediate their chemistry by different processes, demonstrating a convergent evolution of function. But how could two such very different enzymes be recruited to this pathway and be adapted to participate is such a complex biosynthesis? The two enzymes belong to very different protein superfamilies and whilst both are involved in electron transport, the adaptation to a role in oxygenation seems like a very large step. We thus wish to contribute to our knowledge of enzyme catalysed reactions by investigating the exquisite selectivity and efficiency of both CobG and CobZ to cobalamin synthesis. Since both these enzymes are novel, there will be surprises to be discovered on how they catalyse these reactions. We thus believe that this project offers the prospect of insights into enzymatic activity leading to new products and processes. However, we also believe that our investigations will have broader significance and make a positive contribution to the patchwork evolution of pathways, the theory whereby enzymes of broad general specificity are acquired and adapted to specific pathway chemistry. This research consists of fundamental, molecular level studies of the mechanisms of action of enzymes, enzyme substrates and enzymatic systems including multicatalytic complexes. Specifically we will be establishing the molecular mechanisms involved in the biosynthesis of precorrin-3B, including the elucidation of intermediates involved in the reaction. We will establish the kinetics of the enzymatic reaction including the effect of substrates and activators of the enzymes.