Thiamine biosynthesis in Escherichia coli: in vitro reconstitution of the thiazole synthase activity.
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.
Leonardi
Roberta
R
School of Chemistry, University of Southampton, Southampton SO17 1BJ, United Kingdom.
Roach
Peter L
PL
D000241:Adenosine / Q000378:metabolism; D001426:Bacterial Proteins; D013437:Carbon-Sulfur Lyases / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008025:Ligases / Q000737:chemistry; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013466:Sulfurtransferases / Q000502:physiology; D013831:Thiamine / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism / Q000502:physiology
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; C450942:ThiG protein, E coli; C486458:ThiH protein, E coli; D013844:Thiazoles; C411655:thiS protein, E coli; D014443:Tyrosine; D009249:NADP; C005793:thiochrome; D012436:S-Adenosylmethionine; D009713:Nucleotidyltransferases; C411654:ThiF protein, E coli; D013466:Sulfurtransferases; C107110:ThiI protein, bacteria; C491479:thiI protein, E coli; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase; D008025:Ligases; D000241:Adenosine; D003545:Cysteine; D013831:Thiamine