Publications

• Thiamine biosynthesis in Escherichia coli: isolation and initial characterisation of the ThiGH complex.

  Leonardi R., Fairhurst S.A., Kriek M., Lowe D.J., Roach P.L.

  In Escherichia coli, two of the proteins required for the biosynthesis of the thiazole moiety of thiamine (vitamin B(1)) are ThiG and ThiH, encoded as part of the thiCEFSGH operon. In this study, a C-terminally hexahistidine-tagged ThiH (ThiH-His) was expressed in E. coli as a soluble protein from ... >> More

  In Escherichia coli, two of the proteins required for the biosynthesis of the thiazole moiety of thiamine (vitamin B(1)) are ThiG and ThiH, encoded as part of the thiCEFSGH operon. In this study, a C-terminally hexahistidine-tagged ThiH (ThiH-His) was expressed in E. coli as a soluble protein from thiGH-His-tag and thiFSGH-His-tag-bearing plasmids. When isolated under anaerobic conditions, ThiG and ThiH-His co-purify as a large multimeric non-covalent complex. Electron paramagnetic resonance and UV-visible spectroscopy together with iron and sulfide analyses revealed the presence of an iron-sulfur cluster within this complex. << Less

  FEBS Lett. 539:95-99(2003) [PubMed] [EuropePMC]

• Thiamine biosynthesis in Escherichia coli: identification of the intermediate and by-product derived from tyrosine.

  Kriek M., Martins F., Challand M.R., Croft A., Roach P.L.

  Angew. Chem. Int. Ed. 46:9223-9226(2007) [PubMed] [EuropePMC]

• Thiazole synthase from Escherichia coli: an investigation of the substrates and purified proteins required for activity in vitro.

  Kriek M., Martins F., Leonardi R., Fairhurst S.A., Lowe D.J., Roach P.L.

  Thiamine is biosynthesized by combining two heterocyclic precursors. In Escherichia coli and other anaerobes, one of the heterocycles, 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate, is biosynthesized from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine. Genetic evidence has identified thiH ... >> More

  Thiamine is biosynthesized by combining two heterocyclic precursors. In Escherichia coli and other anaerobes, one of the heterocycles, 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate, is biosynthesized from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine. Genetic evidence has identified thiH, thiG, thiS, and thiF as essential for thiazole biosynthesis in E. coli. In this paper, we describe the measurement of the thiazole phosphate-forming reaction using purified protein components. The activity is shown to require four proteins isolated as heterodimers: ThiGH and ThiFS. Reconstitution of the [4Fe-4S] cluster in ThiH was essential for activity, as was the use of ThiS in the thiocarboxylate form. Spectroscopic studies with ThiGH strongly suggested that S-adenosylmethionine (AdoMet) bound to the [4Fe-4S] cluster, which became more susceptible to reduction to the +1 state. Assays of thiazole phosphate formation showed that, in addition to the proteins, Dxp, tyrosine, AdoMet, and a reductant were required. The analysis showed that no more than 1 mol eq of thiazole phosphate was formed per ThiGH. Furthermore, for each mole of thiazole-P formed, 1 eq of AdoMet and 1 eq of tyrosine were utilized, and 1 eq of 5'-deoxyadenosine was produced. These results demonstrate that ThiH is a member of the "radical-AdoMet" family and support a mechanistic hypothesis in which AdoMet is reductively cleaved to yield a highly reactive 5'-deoxyadenosyl radical. This radical is proposed to abstract the phenolic hydrogen atom from tyrosine, and the resultant substrate radical cleaves to yield dehydroglycine, which is required by ThiG for the thiazole cyclization reaction. << Less

  J. Biol. Chem. 282:17413-17423(2007) [PubMed] [EuropePMC]

• Catalytic activity of the anaerobic tyrosine lyase required for thiamine biosynthesis in Escherichia coli.

  Challand M.R., Martins F.T., Roach P.L.

  Thiazole synthase in Escherichia coli is an alphabeta heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the C alpha-C beta bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. This reactive intermediate acts as one of three substrates for the thiazole cy ... >> More

  Thiazole synthase in Escherichia coli is an alphabeta heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the C alpha-C beta bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. This reactive intermediate acts as one of three substrates for the thiazole cyclization reaction catalyzed by ThiG. ThiH is a radical S-adenosylmethionine (AdoMet) enzyme that utilizes a [4Fe-4S](+) cluster to reductively cleave AdoMet, forming methionine and a 5'-deoxyadenosyl radical. Analysis of the time-dependent formation of the reaction products 5'-deoxyadenosine (DOA) and p-cresol has demonstrated catalytic behavior of the tyrosine lyase. The kinetics of product formation showed a pre-steady state burst phase, and the involvement of DOA in product inhibition was identified by the addition of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase to activity assays. This hydrolyzed the DOA and changed the rate-determining step but, in addition, substantially increased the uncoupled turnover of AdoMet. Addition of glyoxylate and ammonium inhibited the tyrosine cleavage reaction, but the reductive cleavage of AdoMet continued in an uncoupled manner. Tyrosine analogues were incubated with ThiGH, which showed a strong preference for phenolic substrates. 4-Hydroxyphenylpropionic acid analogues allowed uncoupled AdoMet cleavage but did not result in further reaction (C alpha-C beta bond cleavage). The results of the substrate analogue studies and the product inhibition can be explained by a mechanistic hypothesis involving two reaction pathways, a product-forming pathway and a futile cycle. << Less

  J Biol Chem 285:5240-5248(2010) [PubMed] [EuropePMC]