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Chemistry

Research project: Roach: Radical AdoMet Proteins

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The catalytic effect and selectivity of many enzymes has been studied in detail and can be explained through the stabilisation of the transition state.

However, some enzymes use very reactive intermediates that open up routes to chemically improbable products. These enzymes not only accelerate the reaction by lowering the transition state energy, but also control the reaction pathway taken, thereby preventing the reactive intermediate from undergoing undesired side reactions. We wish to understand how these enzymes maintain their remarkable product selectivity with very reactive intermediates bound at the active site.

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One group of enzymes that operate via radical mechanisms use the reduction of S-adenosylmethionine to generate methionine and a 5'-deoxyadenosyl radical:

The highly reactive deoxyadenosyl radical is used to remove a hydrogen atom from a second substrate. We have investigated two biosynthetic steps that involve enzymes from this family:

Lipoyl Synthase (LipA)
Thiazole Synthase (ThiGH)
1. Lipoyl Synthase
The insertion of two sulfur atoms by Lipoyl Synthase is one of the most interesting functionalisation reactions in Nature. The reaction is both regio- and stereo- specific, with hydrogen atoms being removed at C6 and C8 of a relatively inert octanoyl chain to allow the introduction of the two sulfur atoms.

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The natural substrates for Lipoyl Synthase include the octanoylated E2 subunit of pyruvate dehydrogenase and LipA is thought to be the source of the sulfur atoms for the reaction.

We have developed an activity assay for Lipoyl Synthase using octanoylated tetra- and tri-peptides as substrate analogues. These octanoyl peptide substrates are useful for studying the chemical mechanism of the reaction and have allowed us to determine the order of sulfur insertion: the first sulfur atom is inserted at C6 and the second sulfur atom is inserted at C8. Currently we are studying the kinetics of the reaction, the products formed from substrate analogues and we are trying to determine the structure of the protein.

Bryant, P., M. Kriek, R. J. Wood and P. L. Roach (2006). "The activity of a thermostable lipoyl synthase from Sulfolobus solfataricus with a synthetic octanoyl substrate." Anal Biochem 351(1): 44-9.
Douglas, P., M. Kriek, P. Bryant and P. L. Roach (2006). "Lipoyl synthase inserts sulfur atoms into an octanoyl substrate in a stepwise manner." Angew Chem Int Ed Engl 45(31): 5197-9.
2. Thiazole Synthase (ThiGH)
The biosynthesis of thiamine is littered with interesting enzymes and mechanisms. One of the most interesting steps is catalysed by the enzyme thiazole synthase which is found in E. coli and other anaerobes. The enzyme was first isolated by us as a heterodimer of two subunits, ThiG and ThiH.

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The ThiH sequence is very similar to other ‘radical-AdoMet' enzymes and this protein uses the 5'-DOA radical to initiate a highly unusual reaction, cleaving the α-β bond of tyrosine. We have recently demonstrated that the ThiGH complex is active in vitro and that the products of tyrosine cleavage by ThiH are p-cresol and dehydroglycine. The ThiG protein utilizes the dehydroglycine to form the thiazole phosphate which is readily incorporated into thiamine.

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We are investigating the mechanism of the enzyme ThiH and our current working hypothesis involves the abstraction of the phenolic hydrogen atom which initiates α-β bond cleavage. We are currently testing the proposed mechanism with substrate analogues and making use of kinetic, spectroscopic and structural studies.

Kriek, M., F. Martins, M. R. Challand, A. Croft and P. L. Roach (2007). "Thiamine biosynthesis in Escherichia coli: identification of the intermediate and by-product derived from tyrosine." Angew Chem Int Ed Engl 46(48): 9223-6.
Kriek, M., F. Martins, R. Leonardi, S. A. Fairhurst, D. J. Lowe and P. L. Roach (2007). "Thiazole synthase from Escherichia coli: an investigation of the substrates and purified proteins required for activity in vitro." J Biol Chem 282(24): 17413-23.
Leonardi, R. and P. L. Roach (2004). "Thiamine biosynthesis in Escherichia coli: in vitro reconstitution of the thiazole synthase activity." J Biol Chem 279(17): 17054-62.
Leonardi, R., S. A. Fairhurst, M. Kriek, D. J. Lowe and P. L. Roach (2003). "Thiamine biosynthesis in Escherichia coli: isolation and initial characterisation of the ThiGH complex." FEBS Lett 539(1-3): 95-9.

Related research groups

Chemical Biology, Diagnostics and Therapeutics
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