Thiazole synthase from Escherichia coli: an investigation of the substrates and purified proteins required for activity in vitro.
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.
Kriek
Marco
M
Chemical Biology Section, School of Chemistry, University of Southampton, Highfield, Southampton, SO17 1BJ, United Kingdom.
Martins
Filipa
F
Leonardi
Roberta
R
Fairhurst
Shirley A
SA
Lowe
David J
DJ
Roach
Peter L
PL
D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015394:Molecular Structure; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013831:Thiamine / Q000096:biosynthesis / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D029968:Escherichia coli Proteins; D021122:Protein Subunits; C450942:ThiG protein, E coli; C486458:ThiH protein, E coli; D013844:Thiazoles; D014443:Tyrosine; D009713:Nucleotidyltransferases; C411654:ThiF protein, E coli; D013831:Thiamine