Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate.
BACKGROUND
We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP), in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a metabolizable carbon source and quickly disappears as soon as the cells receive a carbon source such as glucose. Thus, we hypothesized that AThTP may be a signal produced in response to carbon starvation.


RESULTS
Here we show that, in bacterial extracts, the biosynthesis of AThTP is carried out from thiamine diphosphate (ThDP) and ADP or ATP by a soluble high molecular mass nucleotidyl transferase. We partially purified this enzyme and characterized some of its functional properties. The enzyme activity had an absolute requirement for divalent metal ions, such as Mn2+ or Mg2+, as well as for a heat-stable soluble activator present in bacterial extracts. The enzyme has a pH optimum of 6.5-7.0 and a high Km for ThDP (5 mM), suggesting that, in vivo, the rate of AThTP synthesis is proportional to the free ThDP concentration. When ADP was used as the variable substrate at a fixed ThDP concentration, a sigmoid curve was obtained, with a Hill coefficient of 2.1 and an S0.5 value of 0.08 mM. The specificity of the AThTP synthesizing enzyme with respect to nucleotide substrate is restricted to ATP/ADP, and only ThDP can serve as the second substrate of the reaction. We tentatively named this enzyme ThDP adenylyl transferase (EC 2.7.7.65).


CONCLUSIONS
This is the first demonstration of an enzyme activity transferring a nucleotidyl group on thiamine diphosphate to produce AThTP. The existence of a mechanism for the enzymatic synthesis of this compound is in agreement with the hypothesis of a non-cofactor role for thiamine derivatives in living cells.
Makarchikov
Alexander F
AF
Center for Cellular and Molecular Neurobiology, University of Liège, Liège, Belgium. a_makarchikov@yahoo.com <a_makarchikov@yahoo.com>
Brans
Alain
A
Bettendorff
Lucien
L
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000096:biosynthesis* / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013838:Thiamine Triphosphate / Q000096:biosynthesis* / Q000737:chemistry
D029968:Escherichia coli Proteins; C519454:adenosine thiamine triphosphate; D013838:Thiamine Triphosphate; D008345:Manganese; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; C524661:thiamine diphosphate adenylyl transferase, E coli; D008274:Magnesium
J Biol Chem. 2002 Apr 19;277(16):13771-7
11827967
Nature. 2002 Oct 31;419(6910):952-6
12410317
Cell. 2002 Nov 27;111(5):747-56
12464185
Cell Mol Life Sci. 2003 Jul;60(7):1477-88
12943234
J Biol Chem. 2004 Apr 23;279(17):17142-7
14769791
Nat Chem Biol. 2007 Apr;3(4):211-2
17334376
Anal Biochem. 1976 May 7;72:248-54
942051
Biochem Int. 1989 May;18(5):943-9
2551298
Anal Biochem. 1991 Oct;198(1):52-9
1789432
Nat Chem Biol. 2006 Oct;2(10):529-30
16936719
J Biol Chem. 1976 Jan 10;251(1):193-7
172510