Biosynthesis of 4-methyl-5-(B-hydroxyethyl)thiazole phosphate in Escherichia coli

Leonardi, Roberta (2004) Biosynthesis of 4-methyl-5-(B-hydroxyethyl)thiazole phosphate in Escherichia coli. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Escherichia coli assembles thiamine (vitamin B₁) by coupling 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (Hmp-PP) and 4-methyl-5-(β-hydroxyethyl)thiazole phosphate (Thz-P). These two heterocycles are independently synthesised through pathways that are currently poorly understood. At least six structural proteins, IscS, ThiI and ThiFSGH, participate in the biosynthesis of the intermediate thiazole from tyrosine, 1-deoxy-D-xylulose-5-phosphate (Dxp) and cysteine. As a prerequisite to studying the mechanism of Thz-P biosynthesis, the soluble expression of ThiH, a protein characterised by a marked tendency to form inclusion bodies, was investigated. A C-terminal hexahistidine tagged ThiH (ThiH-His) was successfully expressed in a soluble form from thiGH-His-tag and thiFSGH-His-tag bearing plasmids. Under anaerobic conditions, ThiG and ThiH-His co-purified and were shown to form a large multimeric non-covalent complex. Electron paramagnetic resonance and UV-visible spectroscopies, together with iron and sulphide analyses revealed the presence of an iron-sulphur cluster within this complex. In vitro reconstitution of thiazole synthase activity was also achieved, for the first time, by using cell-free extracts and proteins derived from adenosine-treated E. coli 83.1 cells. The addition of adenosine or adenine to growing cultures of A. aerogenes, S. typhimurium and E. coli has previously been shown to relieve thiamine’s repression of its own biosynthesis and increase the expression levels of the thiamine biosynthetic enzymes. By exploiting this effect, in vitro thiazole synthase activity of cleared lysates or desalted proteins from E. coli 83.1 was shown to be dependent upon the addition of purified ThiGH-His complex, tyrosine (but not cysteine or Dxp) and an as yet unidentified intermediate present in the protein fraction from these cells. The activity was strongly stimulated by the addition of S-adenosylmethionine and NADPH.