The biosynthesis of coenzyme F430 in methanothermobactor thermautotrophicus

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

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Technical Summary

We will elucidate the biosynthesis of coenzyme F430, the yellow tetrapyrrole-derived cofactor required in the terminal step of methanogenesis and which plays a pivotal role in the production of a trillion tons of methane gas per year. Using a cell free system we will determine the intermediates formed between precorrin-2 and coenzyme F430 and attempt to isolate the enzymes. Using a substractive genomic bioinformatics approach we have generated a shortlist of 29 proteins, which is likely to contain all the F430 biosynthetic enzymes. These 29 proteins will be overproduced recombinantly, integrated into a structural genomics programme of research, and also used to investigate their role in the biosynthesis of coenzyme F430.
 
Description The overall aim of the application was to investigate how a prosthetic group called coenzyme F430, which is intricately involved in the annual biogenesis of a billion tonnes of methane gas, is made in a specialised class of bacteria called methanogens. The objectives of the research were to be addressed by using a two-pronged approach, involving both a cell free system to monitor the conversion of uroporphyrinogen III into coenzyme F430 and a recombinant approach where overproduced enzymes were to be tested for their ability to transform intermediates along the pathway.
By studying the genome organisation of methanogens we were able to identify a group of five proteins that are strongly associated with the biosynthesis of F430. We gave these F430 synthesis genes the abbreviation Cfb (for Coenzyme F430 biosynthesis) and provided evidence that they are likely responsible for the transformation of sirohydrochlorin into a 15,173-seco intermediate.
We have shown that CfbA is a nickel chelatase and when co-produced with sirohydrochlorin large amounts of Ni-sirohydrochlorin are produced. CfbE was shown to be an amidase that amidates the a and c side chains of nickel-sirohydrochlorin. The structure of the amidated intermediate was confirmed by NMR. We have also overproduced CfbC and CfbD. These two proteins display similarity to the nitrogenase subunits NirD and NirH and also to the protochlorophyllide reductase subunits BchN and BchD, which are part of the bacteriochlorophyll synthesis pathway. Previous research on nitrogenase activity in M. jannaschii demonstrated that CfbC/D were not involved in nitrogen fixation and the authors suggested that as the proteins also bear a similarity to the protochlorophyllide reductase, whereby they reduce one of the macrocyclic double bonds, that this protein complex may be involved in a similar role in coenzyme F430 biosynthesis. We have shown that it is possible to overproduce both CfbC and CfbD in good yield and, by the coexpression of an isc cluster, also achieve a high yield of Fe-S centre incorporation, as evidenced by EPR. The final protein that we have studied within this main group of Cfb proteins is CfbB, a protein that displays similarity to the ligases associated with peptidoglycan synthesis. Interestingly, when overproduced in E. coli this protein co-purifies with a tightly bound sirohaem. This was somewhat unexpected as the only sirohaem binding proteins known so far are the assimilatory sulfite and nitrite reductases. However, sirohaem is probably binding to CfbB adventitiously as a pseudosubstrate rather than as a prosthetic group. EPR analysis of the CfbB complex revealed that siroheme is present as a low-spin state in the paramagnetic Fe3+ species. In the presence of 1 mM ATP/Mg distinct alterations in the EPR signal intensity occurred without a change in g-value. This could represent ruffling of the sirohaem coordination plane, possibly through ligand induced structural rearrangement within the protein.
Exploitation Route We have identified a number of intermediates (structures determined by NMR and mass spectral analysis) on the F430 biosynthetic route, which has allowed us to propose a new biosynthetic pathway that differs considerably from that previously predicted. We have also located a number of the enzymes involved and determined their activity in vitro. Structural studies have provided molecular detail on the activity of these enzymes. To this end the major goals of the application have been met.

The F430 project also prompted us to look at B12 biosynthesis in M. thermoautotrophicus. The RA1A was able to solve the structure of one of the key enzymes in the cobalamin pathway. The study on the chelatases in M. thermoautotrophicus allowed the RA to identify a novel plant enzyme that she was able to demonstrate is involved in siroheme synthesis, which appears to also contain an Fe-S centre. The results have led to new approaches to the development of synthetic biology applications including the development of multi gene cloning strategies.
Sectors Education,Manufacturing, including Industrial Biotechology
 
Description The research outlined in the application was at the cutting edge of metabolic engineering and synthetic biology in the design of modular and abstractable circuits and as such appealed to two broad beneficiaries - basic scientists trying to understand the nature of fundamental processes within the cell, and applied biotechnologists who are looking for novel findings for exploitation. In terms of basic science this research addressed some simple questions; how are essential coenzymes made within the cell and how can specific pathways be made tractable? The other beneficiaries of the work would include those in the area of applied biotechnology as well as those involved in the development of enhanced productivity. We have outlined an approach that can be used to investigate any biochemical pathway and thus this method may be of interest to those trying to expand pathways for eg secondary metabolites. Our approach also provided new insights into the biosynthesis of modified tetrapyrroles, and helped determine how nature is able to coordinate the resourcing of minerals for biosynthesis. Finally, the data also impacted on bio-engineers, especially those involved in the optimization of biochemical pathways. The research related to how cells can be engineered to help in the elucidation of biochemical pathways and how pathways can then be reconstructed within a new host. This approach is therefore applicable to a broad range of natural products. With an increase in the interest especially of secondary metabolites such an approach is likely to prove popular with chemical biologists and medicinal chemists alike.
First Year Of Impact 2009
Sector Chemicals,Manufacturing, including Industrial Biotechology
Impact Types Societal,Economic