School of Biosciences

Join us in our journey of discovery


profile image for Professor Martin Warren

Professor Martin Warren

Professor of Biochemistry/BBSRC Professorial Fellow

School of Biosciences

  • M.J.Warren@kent.ac.uk

 

Professor Martin Warren joined the School of Biosciences in 2005. He is a member of the Industrial Biotechnology and Synthetic Biology Group and the Centre for Molecular Processing. Martin was born in Northern Ireland, brought up in County Down, and went to Portora Royal School, Enniskillen. He subsequently went to Southampton University where he read Biochemistry as an undergraduate (1981-1984). He stayed on in the Biochemistry Department to do a PhD with Professor Peter Shoolingin-Jordan, which initiated his interest in the genetics and biochemistry of tetrapyrrole biosynthesis.

After completing his PhD studies, he moved in 1989 to Texas A & M University, where he worked as a research associated with Professor Ian Scott FRS on vitamin B12 biosynthesis. In 1991 he took up a lecturing position in the School of Biological Sciences at Queen Mary, University of London, where he stayed until 1995 when he moved to a Senior Lecturer position at the Institute of Ophthalmology, Univeristy College London. He was promoted to Reader of Biochemistry in 1998 but then moved back to the School of Biological Sciences at Queen Mary in 1999 to take up a Personal Chair. In 2005 he moved to the University of Kent, where he is Professor of Biochemistry. In 2007 he was awarded a BBSRC Professorial Fellowship to work on the bioengineering of complex metabolic pathways.

He has published numerous articles on tetrapyrrole biosynthesis and the biochemistry underlying inherited retinopathies, as well as co-authoring a popular book on the link between tetrapyrrole biosynthesis and the madness of George III.

back to top

Also view these in the Kent Academic Repository

Article
Liang, M. et al. (2017). Bacterial microcompartment-directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli. Biotechnology Journal [Online] 12:1600415. Available at: http://doi.org/10.1002/biot.201600415.
Moore, S. et al. (2017). Elucidation of the biosynthesis of the methane catalyst coenzyme F430. Nature [Online]:78-82. Available at: http://dx.doi.org/10.1038/nature21427.
Dailey, H. et al. (2017). Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product. Microbiology and Molecular Biology Reviews [Online] 81:e00048-16. Available at: https://doi.org/10.1128/MMBR.00048-16.
Widner, F. et al. (2016). Total Synthesis, Structure, and Biological Activity of Adenosylrhodibalamin, the Non-Natural Rhodium Homologue of Coenzyme B12. Angewandte Chemie International Edition [Online] 55:11281-11286. Available at: http://doi.org/10.1002/anie.201603738.
Bag, S. et al. (2016). Classification of polyhedral shapes from individual anisotropically resolved cryo-electron tomography reconstructions. BMC Bioinformatics [Online] 17:234-247. Available at: http://doi.org/10.1186/s12859-016-1107-5.
Lee, M. et al. (2016). Employing bacterial microcompartment technology to engineer a shell-free enzyme-aggregate for enhanced 1,2-propanediol production in Escherichia coli. Metabolic Engineering [Online] 36:48-56. Available at: http://doi.org/10.1016/j.ymben.2016.02.007.
Lobo, S. et al. (2016). Desulfovibrio vulgarisCbiKPcobaltochelatase: evolution of a haem binding protein orchestrated by the incorporation of two histidine residues. Environmental Microbiology [Online] 19:106-118. Available at: http://doi.org/10.1111/1462-2920.13479.
Helliwell, K. et al. (2016). Cyanobacteria and Eukaryotic Algae Use Different Chemical Variants of Vitamin B12. Current biology : CB [Online] 26:999-1008. Available at: http://doi.org/10.1016/j.cub.2016.02.041.
Mayer, M. et al. (2016). Effect of bio-engineering on size, shape, composition and rigidity of bacterial microcompartments. Scientific Reports [Online] 6:36899. Available at: http://dx.doi.org/10.1038/srep36899.
Lobo, S. et al. (2015). Staphylococcus aureus haem biosynthesis: characterisation of the enzymes involved in final steps of the pathway. Molecular Microbiology [Online] 97:472-487. Available at: http://doi.org/10.1111/mmi.13041.
Gu, S. et al. (2015). Crystal structure of CobK reveals strand-swapping between Rossmann-fold domains and molecular basis of the reduced precorrin product trap. Scientific Reports [Online] 5:16943-16952. Available at: http://doi.org/10.1038/srep16943.
Palmer, D. et al. (2014). The structure, function and properties of sirohaem decarboxylase - an enzyme with structural homology to a transcription factor family that is part of the alternative haem biosynthesis pathway. Molecular Microbiology [Online] 93:247-261. Available at: http://dx.doi.org/10.1111/mmi.12656.
Moore, S. et al. (2014). Towards a cell factory for vitamin B12 production in Bacillus megaterium: bypassing of the cobalamin riboswitch control elements. New Biotechnology [Online] 31:553-561. Available at: http://dx.doi.org/10.1016/j.nbt.2014.03.003.
Lawrence, A. et al. (2014). FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR). Bioscience Reports [Online] 34:345-355. Available at: http://dx.doi.org/10.1042/BSR20140060.
Bali, S. et al. (2014). Identification and characterization of the 'missing' terminal enzyme for siroheme biosynthesis in -proteobacteria. Molecular Microbiology [Online] 92:153-163. Available at: http://dx.doi.org/10.1111/mmi.12542.
Pang, A. et al. (2014). Structural Insights into Higher Order Assembly and Function of the Bacterial Microcompartment Protein PduA. Journal of Biological Chemistry [Online] 289:22377-22384. Available at: http://dx.doi.org/10.1074/jbc.M114.569285.
Lawrence, A. et al. (2014). Solution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol Bioreactor. ACS Synthetic Biology [Online] 3:454-465. Available at: http://dx.doi.org/10.1021/sb4001118.
Lobo, S. et al. (2014). Characterisation of Desulfovibrio vulgaris haem b synthase, a radical SAM family member. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics [Online] 1844:1238-1247. Available at: http://dx.doi.org/10.1016/j.bbapap.2014.03.016.
Bali, S. et al. (2014). Recent advances in the biosynthesis of modified tetrapyrroles: the discovery of an alternative pathway for the formation of heme and heme d 1. Cellular and Molecular Life Sciences [Online] 71:2837-2863. Available at: http://dx.doi.org/10.1007/s00018-014-1563-x.
Moore, S. et al. (2013). Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12). Proceedings of the National Academy of Sciences [Online] 110:14906-14911. Available at: http://dx.doi.org/10.1073/pnas.1308098110.
Azim, N. et al. (2013). Crystallization and preliminary X-ray characterization of the tetrapyrrole-biosynthetic enzyme porphobilinogen deaminase from Bacillus megaterium. Acta Crystallographica Section F Structural Biology and Crystallization Communications [Online] 69:906-908. Available at: http://dx.doi.org/10.1107/S1744309113018526.
Moore, S. et al. (2013). Characterization of the enzyme CbiH60 involved in anaerobic ring contraction of the cobalamin (vitamin B12) biosynthetic pathway. Journal of Biological Chemistry [Online] 288:297-305. Available at: http://dx.doi.org/10.1074/jbc.M112.422535.
Collins, H. et al. (2013). Bacillus megaterium has both a functional BluB protein required for DMB synthesis and a related flavoprotein that forms a stable radical species. PLoS ONE [Online] 8:e55708. Available at: http://dx.doi.org/10.1371/journal.pone.0055708.
Saha, K. et al. (2012). Characterization of the evolutionarily conserved iron-sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana. Biochemical Journal [Online] 444:227-237. Available at: http://dx.doi.org/10.1042/BJ20111993.
Moore, S. and Warren, M. (2012). The anaerobic biosynthesis of vitamin B12. Biochemical Society Transactions [Online] 40:581-586. Available at: http://dx.doi.org/10.1042/BST20120066.
Deery, E. et al. (2012). An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis. Nature Chemical Biology [Online] 8:933-940. Available at: http://dx.doi.org/10.1038/nchembio.1086.
Kazamia, E. et al. (2012). Mutualistic interactions between vitamin B12 -dependent algae and heterotrophic bacteria exhibit regulation. Environmental Microbiology [Online] 14:1466-1476. Available at: http://dx.doi.org/10.1111/j.1462-2920.2012.02733.x.
Warman, A. et al. (2012). Characterization of Cupriavidus metallidurans CYP116B1- a thiocarbamate herbicide oxygenating P450-phthalate dioxygenase reductase fusion protein. FEBS Journal [Online] 279:1675-1693. Available at: http://dx.doi.org/10.1111/j.1742-4658.2012.08543.x.
Pang, A., Warren, M. and Pickersgill, R. (2011). Structure of PduT, a trimeric bacterial microcompartment protein with a 4Fe4S cluster-binding site. Acta Crystallographica Section D-Biological Crystallography [Online] 67:91-96. Available at: http://dx.doi.org/10.1107/S0907444910050201.
Romao, C. et al. (2011). Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization. Proceedings of the National Academy of Sciences of the United States of America [Online] 108:97-102. Available at: http://dx.doi.org/10.1073/pnas.1014298108.
Hansson, M. et al. (2011). Bacterial ferrochelatase turns human: Tyr13 determines the apparent metal specificity of Bacillus subtilis ferrochelatase. JBIC Journal of Biological Inorganic Chemistry [Online] 16:235-242. Available at: http://dx.doi.org/10.1007/s00775-010-0720-4.
Bali, S. et al. (2011). Molecular hijacking of siroheme for the synthesis of heme and d1 heme. Proceedings of the National Academy of Sciences of the United States of America [Online] 108:18260-18265. Available at: http://dx.doi.org/10.1073/pnas.1108228108.
Biedendieck, R. et al. (2010). Metabolic engineering of cobalamin (vitamin B12) production inBacillus megaterium. Microbial Biotechnology [Online] 3:24-37. Available at: http://dx.doi.org/10.1111/j.1751-7915.2009.00125.x.
Bali, S., Warren, M. and Ferguson, S. (2010). NirF is a periplasmic protein that binds d1 heme as part of its essential role in d1 heme biogenesis. FEBS Journal [Online] 277:4944-4955. Available at: http://dx.doi.org/10.1111/j.1742-4658.2010.07899.x.
Kerfeld, C. et al. (2010). Characterisation of PduS, the pdu Metabolosome Corrin Reductase, and Evidence of Substructural Organisation within the Bacterial Microcompartment. PLoS ONE [Online] 5:e14009. Available at: http://dx.doi.org/10.1371/journal.pone.0014009.
Seyedarabi, A. et al. (2010). Cloning, purification and preliminary crystallographic analysis of cobalamin methyltransferases fromRhodobacter capsulatus. Acta Crystallographica Section F Structural Biology and Crystallization Communications [Online] 66:1652-1656. Available at: http://dx.doi.org/10.1107/S1744309110042910.
Storbeck, S. et al. (2010). A Novel Pathway for the Biosynthesis of Heme in Archaea: Genome-Based Bioinformatic Predictions and Experimental Evidence. Archaea [Online] 2010:1-15. Available at: http://dx.doi.org/10.1155/2010/175050.
Brindley, A. et al. (2010). NirJ, a radical SAM family member of the d1 heme biogenesis cluster. FEBS Letters [Online] 584:2461-2466. Available at: http://dx.doi.org/10.1016/j.febslet.2010.04.053.
Schroeder, S. et al. (2009). Demonstration That CobG, the Monooxygenase Associated with the Ring Contraction Process of the Aerobic Cobalamin (Vitamin B12) Biosynthetic Pathway, Contains an Fe-S Center and a Mononuclear Non-heme Iron Center. Journal of Biological Chemistry [Online] 284:4796-4805. Available at: http://dx.doi.org/10.1074/jbc.M807184200.
Zajicek, R. et al. (2009). d1 haem biogenesis - assessing the roles of three nir gene products. FEBS Journal [Online] 276:6399-6411. Available at: http://dx.doi.org/10.1111/j.1742-4658.2009.07354.x.
Lotierzo, M. et al. (2009). Ironsulfur cluster dynamics in biotin synthase: A new [2Fe2S]1+ cluster. Biochemical and Biophysical Research Communications [Online] 381:487-490. Available at: http://dx.doi.org/10.1016/j.bbrc.2009.02.089.
Lobo, S. et al. (2009). Functional characterization of the early steps of tetrapyrrole biosynthesis and modification in Desulfovibrio vulgaris Hildenborough. Biochemical Journal [Online] 420:317-325. Available at: http://dx.doi.org/10.1042/BJ20090151.
Lobo, S. et al. (2008). Two distinct roles for two functional cobaltochelatases (CbiK) in Desulfovibrio vulgaris Hildenborough. Biochemistry [Online] 47:5851-5857. Available at: http://dx.doi.org/http://pubs.acs.org/doi/abs/10.1021/bi800342c.
Sriramulu, D. et al. (2008). Lactobacillus reuteri DSM 20016 produces cobalamin-dependent diol dehydratase in metabolosomes and metabolizes 1,2-propanediol by disproportionation. Journal of Bacteriology [Online] 190:4559-4567. Available at: http://dx.doi.org/10.1128/jb.01535-07.
Lawrence, A. et al. (2008). Identification, characterization, and structure/function analysis of a corrin reductase involved in adenosylcobalamin biosynthesis. Journal of Biological Chemistry [Online] 283:10813-10821. Available at: http://dx.doi.org/10.1074/jbc.M710431200.
Parsons, J. et al. (2008). Biochemical and Structural Insights into Bacterial Organelle Form and Biogenesis. Journal of Biological Chemistry [Online] 283:14366-14375. Available at: http://dx.doi.org/10.1074/jbc.M709214200.
Schubert, H. et al. (2008). Structure and function of SirC from Bacillus megaterium: a metal-binding precorrin-2 dehydrogenase. Biochemical Journal 415:257-263.
Wilkie, S. et al. (2008). Disease mechanism for retinitis pigmentosa (RP11) caused by missense mutations in the splicing factor gene PRPF31. Molecular Vision 14:683-690.
Holliday, G. et al. (2007). Evolution of enzymes and pathways for the biosynthesis of cofactors. Natural Product Reports [Online] 24:972-987. Available at: http://www.rsc.org/Publishing/Journals/NP/article.asp?doi=b703107f.
Mendel, R. et al. (2007). Metal and cofactor insertion. Natural Product Reports [Online] 24:963-971. Available at: http://dx.doi.org/10.1039/b703112m.
Rébeillé, F. et al. (2007). Roles of vitamins B5, B8, B9, B12 and molybdenum cofactor at cellular and organismal levels. Natural Product Reports [Online] 24:949-962. Available at: http://dx.doi.org/10.1039/B703104C.
Frank, S. et al. (2007). Elucidation of substrate specificity in the cobalamin (vitamin B12) biosynthetic methyltransferases. Structure and function of the C20 methyltransferase (CbiL) from Methanothermobacter thermautotrophicus. Journal of Biological Chemistry [Online] 282:23957-23969. Available at: http://dx.doi.org/10.1074/jbc.M703827200.
Marquet, A. et al. (2007). Iron-sulfur proteins as initiators of radical chemistry. Natural Product Reports [Online] 24:1027-1040. Available at: http://www.rsc.org/publishing/journals/NP/article.asp?doi=b703109m.
Layer, G. et al. (2006). The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN. Journal of Biological Chemistry [Online] 281:15727-34. Available at: http://dx.doi.org/10.1074/jbc.M512628200.
Warren, M. (2006). Finding the final pieces of the vitamin B12 biosynthetic jigsaw. Proc Natl Acad Sci U S A [Online] 103:4799-800. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16567660 .
Cox, T. et al. (2005). King George III and porphyria: an elemental hypothesis and investigation. Lancet [Online] 366:332-335. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16039338.
Frank, S. et al. (2005). Anaerobic synthesis of vitamin B12: characterization of the early steps in the pathway. Biochemical Society Transactions [Online] 33:811-814. Available at: http://dx.doi.org/10.1042/BST0330811.
McGoldrick, H. et al. (2005). Identification and characterization of a novel vitamin B12 (cobalamin) biosynthetic enzyme (CobZ) from Rhodobacter capsulatus, containing flavin, heme, and Fe-S cofactors. Journal of Biological Chemistry [Online] 280:1086-1094. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15525640.
Croft, M. et al. (2005). Algae acquire vitamin B12 through a symbiotic relationship with bacteria. Nature [Online] 438:90-93. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16267554.
Vevodova, J. et al. (2004). Structure/function studies on a S-adenosyl-L-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis. Journal of Molecular Biology [Online] 344:419-33. Available at: http://dx.doi.org/10.1016/j.jmb.2004.09.020 .
Erskine, P. et al. (2003). X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase. Biochemical Journal [Online] 373:733-8. Available at: http://dx.doi.org/10.1042/BJ20030513.
Leech, H. et al. (2003). Characterization of the cobaltochelatase CbiXL: evidence for a 4Fe-4S center housed within an MXCXXC motif. Journal of Biological Chemistry [Online] 278:41900-7. Available at: http://dx.doi.org/10.1074/jbc.M306112200.
Wilkie, S. et al. (2002). Characterisation of two genes for guanylate cyclase activator protein (GCAP1 and GCAP2) in the Japanese pufferfish, Fugu rubripes. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression [Online] 1577:73-80. Available at: http://dx.doi.org/10.1016/S0167-4781(02)00413-X .
Schubert, H. et al. (2002). The structure of Saccharomyces cerevisiae Met8p, a bifunctional dehydrogenase and ferrochelatase. EMBO Journal [Online] 21:2068-2075. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11980703.
Deery, E. et al. (2002). Disease mechanism for retinitis pigmentosa (RP11) caused by mutations in the splicing factor gene PRPF31. Human Molecular Genetics [Online] 11:3209-3219. Available at: http://dx.doi.org/10.1093/hmg/11.25.3209.
Roessners, C. et al. (2002). Isolation and characterization of 14 additional genes specifying the anaerobic biosynthesis of cobalamin (vitamin B12) in Propionibacterium freudenreichii (P. shermanii). Microbiology 148:1845-53.
Schubert, H. et al. (2001). Optimization of Met8p crystals through protein-storage buffer manipulation. Acta Crystallographica Section D-Biological Crystallography [Online] 57:867-869. Available at: http://dx.doi.org/10.1107/S0907444901004619.
Wilkie, S. et al. (2001). Identification and functional consequences of a new mutation (E155G) in the gene for GCAP1 that causes autosomal dominant cone dystrophy. American Journal Humman Genetics 69:471-80.
Roper, J. et al. (2000). The enigma of cobalamin (Vitamin B12) biosynthesis in Porphyromonas gingivalis. Identification and characterization of a functional corrin pathway. Journal of Biological Chemistry [Online] 275:40316-23. Available at: http://dx.doi.org/10.1074/jbc.M007146200 .
Book section
Lobo, S., Warren, M. and Saraiva, L. (2012). Sulfate-reducing bacteria reveal a new branch of tetrapyrrole metabolism. in: Advances in Microbial Physiology. Elsevier Ltd, pp. 267-295. Available at: http://dx.doi.org/10.1016/B978-0-12-394423-8.00007-X.
Hunt, D. et al. (2004). Dominant cone and cone-rod dystrophies: functional analysis of mutations in retGC1 and GCAP1. in: Bock, G., Chader, G. and Goode, J. eds. Retinal Dystrophies: Functional Genomics to Gene Therapy. John Wiley & Sons, pp. 37-49. Available at: http://dx.doi.org/10.1002/0470092645.ch4.
Showing 71 of 86 total publications in KAR. [See all in KAR]
back to top

Warren’s research envelops areas of chemistry, biology and history. His interests are firmly embedded in the biosynthesis and biology of the pigments of life, encompassing molecules such as heme, chlorophyll, vitamin B12, siroheme, coenzyme F430 and heme d1. With Dr Evelyne Deery he was one of the first to use synthetic biology as a means to probe biosynthetic pathways through the reconstruction of the whole cobalamin (vitamin B12) pathway in E. coli, an organism that does not possess the ability to make this molecule de novo. More recently he has elucidated a novel alternative heme biosynthesis pathway that is present in both archaea and sulphate reducing bacteria, where siroheme is hijacked as a substrate – a rare example of where one prosthetic group is cannibalised for the synthesis of another. His interest in vitamin B12 chemistry led to a study of how cobalamin is used for propanediol utilisation (pdu) in some bacteria, where remarkably the metabolic process is sequestered within a proteinaceous organelle called a bacterial micrcompartment, one of the largest protein-based complexes found in nature. Here, again, the entire pdu operon was cloned into E. coli and shown to orchestrate the construction of functional recombinant pdu microcompartments. Subsequently, empty organelles have been engineered through the targeting of new proteins and processes to these bodies. His interest in heme metabolism has also contributed to a new subject of scientific historiography. Here, he has studied George III, America’s last king, whose madness has been attributed to attacks of porphyria, an inborn error of heme metabolism. Through research in the Royal Archives in Windsor Castle and DNA analysis of some of the King’s descendants, coupled with a forensic analysis of the King’s hair, he has shown that it is likely that the much-maligned monarch suffered with variegate porphyria, worsened by the administration of antimony-based medication that was contaminated with arsenic.

BBSRC Funded research projects:

Bioengineering of complex metabolic pathways - Professorial fellowship 2008-2012

Vitamins are essential nutrients required by humans to complete their diet, and by definition are not made within their own body. Plants and vegetables are good sources of many vitamins. However, plants are missing one vitamin from their complement / vitamin B12. This is because plants neither make nor require vitamin B12 within their metabolism. In fact, vitamin B12 is unique among the vitamins in that it is the only vitamin whose synthesis is restricted solely to bacteria, as the ability to make this nutrient never successfully made the prokaryote to eukaryote transition. The 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 systems 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 increasing the levels of vitamin B12 in the diet.
In this research programme we wish to explore the limitations and consequences of engineering complex metabolic pathways into different organisms, taking advantage of the latest developments and technologies in metabolic engineering. We plan to take the genetic software that allows bacteria to make vitamin B12 and transfer it into bacteria that are unable to make B12, yeast and a higher plant, thereby conferring upon these organisms the ability to make this essential nutrient.
For bacteria, we wish to explore how the pathway can be enhanced for maximum vitamin production. Will increased levels of certain enzymes give increased metabolic flux, or will substrate bioavailabilty be limiting? These are questions that we will address in this application. For engineering into yeast, we have to look at a complex cloning procedure that ensures the genes have separate promoters and regulatory regions. For plants, we will take advantage of the fact that certain organelles within the plant, called plastids, have their own genetic material and in essence behave like bacteria within the plant. We will integrate the DNA from a bacterial species into the plastid and then monitor the level of the vitamin made during the growth of the plant. In fact, we will make a number of variants, of increasing complexity, to see how the plant is able to cope with this genetic modification.
This project 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. The results of the project will provide knowledge that can be used to develop new technologies and products for agriculture and bioremediation.

Unravelling the remarkable synthesis and mechanisms involved in the biogenesis of heme and heme d1 from siroheme

Co-applicants: Prof Stuart Ferguson (Oxford), Dr Mark Howard and Dr Stephen Rigby (Manchester) 2012-2014

This research aims to have a major impact on our understanding of the synthesis and evolution of a related family of heme-like molecules, and will result in a comprehensive addition to text book sections dealing with porphyrin metabolism. Hemes belong to a family of essential life pigment involved in many basic respiratory processes. This project aims to decipher a twenty-year mystery by unravelling the pathway leading to their synthesis. Specifically we aim to elucidate how heme and heme d1 are made in the Archaea and a number of eubacteria.  Heme was previously thought to be synthesized along a single pathway from a compound called uroporphyrinogen III by a series of enzymes that modify the acidic side chains, oxidize the macrocycle and insert ferrous iron. However, we have recently demonstrated that heme and a structurally related macrocycle, heme d1, can be made by a completely novel pathway that involves the cannibalism of another modified tetrapyrrole, a compound called siroheme, the prosthetic group of sulphite and nitrite reductase.
There are many novel features to this project that make it compelling. The transformation of siroheme into heme and heme d1 requires a number of radical SAM enzymes coupled with a decarboxylase, involving some highly unusual steps that are unprecedented in biological chemistry. The aim of this research is to understand in molecular detail the step-by-step synthesis of hemes along this pathway.
Although the d1 heme is only found in the cytochrome cd1, the importance of this enzyme is becoming ever more evident as it vital for the environmentally important ANAMMOX process whereby ammonia is oxidised by nitrite under anaerobic conditions and has recently been implicated in playing a key role in the anaerobic oxidation of methane. It is believed that d1 heme has been adopted for respiratory nitrite reduction because it is tuned to permit nitrite reduction only as far as nitric oxide and not to allow the onward reduction to ammonia that is catalysed by siroheme. The discovery that siroheme is also a precursor for d1 synthesis therefore raises many interesting evolutionary and regulatory questions.
Overall, the outcome of this research will result in the composition of a molecular overture of events for a new heme synthesis route, explain more clearly the relationship between heme and siroheme and provide a neodarwinistic understanding of complex biosynthetic pathways. It will also help maintain the UK at the cutting edge of biomolecular science research and maintain the position our groups have as world leaders in this field.

.

Enzymes as traps in the elucidation of complex biochemical pathways

Co-applicants Prof Richard Pickersgill (Queen Mary), Prof Mike Geeves and Dr Mark Howard 2012-2015

In this research project we will apply a method that will allow a step-change in our ability to study complex biochemical pathways, provide molecular detail on fascinating enzyme mechanisms and to rewrite the metabolic control of pathways involving labile intermediates. The elucidation of biochemical pathways is a challenging area that is often complicated by low levels of inherently unstable metabolic intermediates. We have developed a method that allows for the isolation of enzyme-bound metabolites, permitting their characterisation and thereby providing an opportunity to gain atomic resolution of a number of fascinating enzyme-mediated transformations. The research is based on the finding that in some biochemical pathways the product of one reaction is passed directly onto the next in a process known as substrate channelling. Key to this is a tight association between an enzyme and its product, which allows for the isolation of highly stable enzyme-product complexes. We will exploit these properties to unravel the mysteries surrounding the biosynthesis of vitamin B12 (cobalamin).  By using His-tagged enzymes of the pathway it is now possible to isolate many of the hitherto ephemeral intermediates, trapped and stabilised on the tagged enzymes as tightly bound enzyme-product complexes. Characterisation of these intermediates will allow the complete elucidation of the corrin pathway. Moreover, a combination of enzymology and X-ray crystallography will permit a detailed understanding of the mechanism of the enzymes that mediate the synthesis of the corrin framework, including the ring contraction process that involves the extrusion of an integral carbon atom in a reaction that has no parallel in nature. Our preliminary data is consistent with the B12 pathway operating by direct metabolite channelling. We outline experiments to investigate this further and to determine whether enzyme rather than substrate concentration controls this metabolic process.

Synthetic biology approaches to compartmentalisation in bacteria and the construction onovel bioreactors

Co-applicants Dr Dan Mulvihill, Prof Richard Pickersgill (Queen Mary), Dr Ian Blomfield 2010-2013

Synthetic biology has emerged from the development of techniques that allow for significant alterations in the metabolism/physiology of the cell to produce desirable products, including drugs, chemicals, vitamins, biofuels etc. Ultimately synthetic biology could lead to the construction of new metabolic pathways and even new life forms. A major advance in the area of synthetic biology would be the ability to make compartments in the cell to house specific metabolic processes. The development of such bespoke organelles would allow for greater control and regulation of the process, detaching the encased process from any negative influences within cellular metabolism. We have demonstrated that it is possible to synthesise such organelles in E. coli by the metabolic engineering of the 1,2-propanediol utilizing (pdu) operon, a complex that contains around 20,000 polypeptide subunits and has a molecular mass of approximately 300 mDa. More recently, we have shown that it is possible to make empty bacterial microcompartments (BMCs) by the coordinated overproduction of 5 gene products that compose the shell (outer casing) of the organelle. We have also demonstrated that proteins normally encased within the BMC are targeted to this empty vesicle and that other non-vesicle proteins can be incorporated to the BMC by fusing them onto target proteins. By labelling the proteins with GFP it has been possible to undertake live cell imaging of the organelles and evidence for movement between organelles via filaments has been observed.
We now wish to extend this study by directing specific enzymes to the BMC in order to make bespoke bioreactors. Such an approach will provide insights into the metabolic advantage of compartmentalisation. Engineering of the shell proteins will be undertaken to help in the rational design of a semi permeable shell with a broader substrate specificity. The intramolecular orientation of the shell proteins within the organelle will be investigated by a combination of antibodies and proteolytic processing. Detail on the intermolecular arrangement of shell proteins within the BMC will be obtained from studies using a range of different GFP-shell protein fusions and the analysis of the BMC by FRET. In this way we will be able to generate a model of the organelle and this, in combination with a number of other experimental approaches, will permit an investigation into how proteins are targeted and incorporated into the BMC. The rate of protein exchange in the organelle and the order of incorporation into the organelle will be defined not only using live cell imaging by also by employing FRAP.  Detail on how the organelle is held within the cell will be investigated using a range of directed cell cytoskeleton mutants. The role of a Ras-type GTPase, PduV, in organelle dynamics will also be probed.

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

Co-applicants Prof Gerald Richter (Cardiff), Prof Mike Geeves, Dr Gary Robinson 2010-2012

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 anaemia 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 vegetarians –(where the absence of B12 in plants make vegetarians, especially vegans, susceptible to deficiency), the elderly  (up to 30% of people over 70 have B12 deficiency and require supplements and, in some cases, intravenous injections of B12) and unborn babies (where 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 correct version of the vitamin, and include certain probiotic bacteria that are sold in liquid cultures and 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 research 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.

 

back to top

Year 2

  • BI520 Metabolism and Metabolic Disease
  • BI521 Metabolism and Metabolic Regulation
  • BI629 Proteins
back to top
back to top

Post-docs:

  • Dr Evelyne Deery
  • Dr Andrew Lawrence
  • Dr Dimitrios Ladakis
  • Mr David Palmer
  • Dr Minghzi Liang

PhD students:

back to top

Enquiries: Phone: +44 (0)1227 823743

School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ

Last Updated: 15/01/2016