Mechanism of dimethylenzimidazole (DMB) synthesis and the metabolic engineering of a dietary useful form of cobalamin in Lactobacillus

Lead Research Organisation: University of Kent

Abstract

Vitamin B12, also known as cobalamin, is required for two essential enzymes in humans, methylmalonyl CoA mutase and methionine synthase and the lack of B12 prevents these enzymes functioning and results in pernicious anaemia. Although a form of the disease can occur in children, pernicious anemia usually does not appear before the age of 30. The average age at diagnosis is 60. The disease occurs in all racial groups, but occurs most often in people of Scandinavian or Northern European descent. The disease may be manifest as a lack of vitamin B12 and / or an inability to absorb B12, both of which may be treated by vitamin B12 administration. Vitamin B12 supplements can be given but 1000 microg / day are required. We ordinarily obtain vitamin B12 from our diet, particularly from meat and dairy products but not from fruit and vegetables, which do not contain this essential nutrient. Vitamin B12 deficiency results in a plethora of clinical manifestations including hematologic, neurologic and psychiatric disorders. Those people at risk of vitamin B12 deficiency include: 1. Vegetarians - the absence of B12 in plants make vegetarians, especially vegans, susceptible to deficiency. 2. Elderly - up to 30% of people over 70 have B12 deficiency and require supplements and, in some cases, intravenous injections of B12 3. Unborn babies - B12 is a contributory risk factor in neural tube defects in unborn babies. There is thus a need to provide suitable foodstuffs with elevated cobalamin levels. Some countries are even contemplating adding B12 to flour in the same way that folate is already added. Vitamin B12 is unique amongst the vitamins, being manufactured only by prokaryotes. It is produced commercially by fermentation using specific strains that have been selected for high B12 levels. Many strains and production methodologies have been used in the past but the principal methodology employed involves the co-feeding of a component of the vitamin called 5,6-dimethyl benzimidazole (DMB) to the culture medium. The synthesis and attachment of DMB is often rate-limiting in the biosynthesis of the pathway. Interestingly, as any trip to supermarket, health food shop or browse of the internet will demonstrate vitamin B12 can be purchased in a range of forms. However, many of these forms are useless as they do not contain the the correct version of the vitamin, and include: 1. Certain probiotic bacteria that are sold in liquid cultures 2. Spirulina extracts, which are marketed as a source of vitamin B12 in the belief that, as cyanobacteria, they produce large amounts of the vitamin. In these cases the proposed 'vitamin B12' is not cobalamin but is pseudocobalamin which, although structurally similar, is unable to be used by humans. The difference between the two compounds is the identity of the lower ligand with pseudocobalamin containing adenine and cobalamin containing 5,6-dimethylbenzamide (DMB). Expertise at Kent and elsewhere has contributed to our understanding of the synthetic pathway, leading to the characterisation of the protein responsible for the synthesis of DMB, enabling the production of true vitamin B12. The gene encoding this critical step (bluB) is present in certain bacterial strains but absent in others. The overall strategy in the current proposal is to understand how DMB is made by the enzyme BluB. Subsequently, bluB will be transferred into a pseudocobalamin-producing probiotic strain that has GRAS (Generally Regarded As Safe) status. This is the first attempt to create a genetically engineered bacterium for use in probiotic formulations with the capability of providing sustainable vitamin B12 production in vivo.

Technical Summary

Vitamin B12 (cobalamin) represents one of the most complex small molecules made ubiquitously in living systems. This structural complexity is underpinned by an equally elaborate and intricate biochemical pathway that requires somewhere around thirty enzyme-mediated steps. One of these steps requires the transformation of the isoalloxazine ring of FMN into dimethylbenzimidazole (DMB), the base that is found as the lower axial cobalt ligand in the final B12 molecule. This 'molecular cannibalism' of one coenzyme to form another is unique to cobalamin biosynthesis. The transformation of FMN into DMB requires the breaking and making of a large number of bonds yet this amazing transformation is catalysed by a comparatively small (24 kDa) enzyme called BluB. The overall aim of this project is to gain some molecular insights into how this enzyme is able to mediate such a seemingly complex process. We will investigate this using a chemical biological approach, by using flavo coenzyme analogues that will block the reaction part way through the process. This will be complemented by molecular enzymological and structural approaches that will provide molecular detail on the individual steps of the reaction process. Many bacteria do not make DMB but instead incorporate adenine into the vitamin B12 molecule to give pseudocobalamin, which although structurally similar is unable to be used by humans. In the final part of the grant we plan to use a synthetic biology approach and engineer the terminal part of the cobalamin pathway into a pseudocobalamin-producing strain of Lactobacillus, thereby giving it the ability to make the nutritionally important form of the vitamin. This will be the first attempt to generate a metabolically engineered bacterium for use in probiotic formulations.
 
Description The grant concerned the synthesis of the unusual base dimethylbenzimidazole (DMB) that is found in the lower nucleotide of vitamin B12. The grant investigated how DMB is made from reduced flavin. We demonstrated that the transformation of reduced flavin into dimethylbenzimidazole proceeds via the formation of a 4a-peroxyflavin intermediate after the initial reaction with molecular oxygen. We also characterized BluB from Bacillus megaterium and showed that this enzyme has a much lower activity, reflecting the fact that this organism grows in an aerobic environment.

We solved the structure of the enzyme with a range of different flavins in different oxidation states. The structures reveal a partial fragmentation of the substrate indicating that the reaction has been caught part way through the self-destructive process. We also cloned the bluB gene from R. capsulatus into lactobacillus with a view to studying if the synthesis of dimethylbenzimidazole results in the biosynthesis of cobalamin rather than pseudocobalamin.

The main objectives of the grant have been met. These have included:
1. The synthesis of a range of modified riboflavins including the 1, 3 and 5 deaza derivatives.
2. The derivatives have been tested for activity within the enzyme, after conversion into their FMN forms. Unfortunately all the derivatives acted as inhibitors rather than substrates indicating that they did not bind in the active site in a productive manner to allow interaction with molecular oxygen.
3. The successful characterisation of the enzyme by molecular enzymology. Stopped flow analysis revealed the initial formation of a peroxyflavin intermediate.
4. The structure determination of the enzyme by X-ray crystallography, with structures of the enzyme containing a range of different flavins.
Exploitation Route The enzyme catalyses a remarkable reaction involving the making and breaking of C-C and C-N bonds. The unique chemistry catalysed by this comparatively small enzyme still requires further elucidation to understand how all the bond making and breaking are accommodated within the active site. Te ability to make DMB is important for cobalamin producers - and making larger amounts of DMB is known to have a significant effect on the overall production of cobalamin. There is significant interest in being able to produce more DMB that can be fed into the cobalamin biosynthetic pathway.
Sectors Chemicals,Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The engineering of enzymes such as BluB for enhanced production of DMB is being investigated by several companies. The ability to make this compound, and especially to feed it directly into a cobalamin biosynthetic pathway, will have significant benefits to those involved in vitamin production. Other research has shown that increased levels of DMB e.g. provided to diary cows also results in higher yields of vitamin B12 in milk - thus demonstrating further the importance of the provision of this base to the vitamin biosynthetic pathway. Therefore, this research is underpinning important science relating to improved health and well-being. We are looking at how certain bacteria can be used to help promote cobalamin production and whether other bacteria that produce cobalamin-analogues can be modified so that they also produce cobalamin. This will be of particular relevance to bacteria that occupy the small intestine.
First Year Of Impact 2011
Sector Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology