Synthesis and accumulation of thiamin triphosphate in Escherichia coli cells expressing chicken cytosolic adenylate kinase.
To examine whether cytosolic adenylate kinase (AK1, EC 2.7.4.3) catalyzes synthesis of thiamin triphosphate (TTP) in vivo, chicken AK1 was expressed in Escherichia coli, and cellular AK1 activity and TTP content were determined. E. coli harboring the vector plasmid was used as a control. Chicken AK1 was expressed in the producer strain at a high level (83 U/mg protein) even without inducers, and this expression was doubled (153 U/mg protein) by beta-D-isopropylthiogalactopyranoside (IPTG). TTP was accumulated in the producer cells at a high level (5.7 nmol/g dry weight) without IPTG and this was also doubled (10.2 nmol/g dry weight) by IPTG. TTP content in the control strain was very low (0.2-0.9 nmol/g dry weight) and was unaffected by IPTG. Neither bacterial growth curve nor cellular content of AMP, ADP, ATP, thiamin diphosphate or total thiamin (sum of thiamin and its phosphate esters) was different between the producer and the control strains. These results indicate that chicken AK1 expressed in E. coli catalyzed the synthesis and accumulation of TTP within the bacterial cells.
Shioda
T
T
Department of Biochemistry, Hiroshima University School of Medicine, Japan.
Egi
Y
Y
Yamada
K
K
Yamada
M
M
Nakazawa
A
A
Kawasaki
T
T
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002474:Cell-Free System; D002645:Chickens; D003600:Cytosol / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D013838:Thiamine Triphosphate / Q000096:biosynthesis* / Q000378:metabolism
D013838:Thiamine Triphosphate; D007544:Isopropyl Thiogalactoside; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase