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J. Biol. Chem., Vol. 279, Issue 17, 17054-17062, April 23, 2004

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Thiamine Biosynthesis in Escherichia coli

IN VITRO RECONSTITUTION OF THE THIAZOLE SYNTHASE ACTIVITY^*

Roberta Leonardi and Peter L. Roach

From the School of Chemistry, University of Southampton, Southampton SO17 1BJ, United Kingdom

The biosynthesis of thiamine in Escherichia coli requires the formation of an intermediate thiazole from tyrosine, 1-deoxy-D-xylulose-5-phosphate (Dxp), and cysteine using at least six structural proteins, ThiFSGH, IscS, and ThiI. We describe for the first time the reconstitution of thiazole synthase activity 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 Aerobacter aerogenes, Salmonella typhimurium, and E. coli has been shown previously to relieve the repression by thiamine of its own biosynthesis and increase the expression levels of the thiamine biosynthetic enzymes. By exploiting this effect, we show that the in vitro thiazole synthase activity of cleared lysates or desalted proteins from E. coli 83-1 cells is dependent upon the addition of purified ThiGH-His complex, tyrosine (but not cysteine or 1-deoxy-D-xylulose-5-phosphate), and an as yet unidentified intermediate present in the protein fraction from these cells. The activity is strongly stimulated by the addition of S-adenosylmethionine and NADPH.

Received for publication, November 20, 2003 , and in revised form, January 20, 2004.

^* This work was supported by a University Research Fellowship from the Royal Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: School of Chemistry, University of Southampton, University Rd., Highfield, SO17 1BJ Southampton, UK. Tel.: 44-2380-59591; Fax: 44-2380-596805; E-mail: plr2{at}soton.ac.uk.

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