Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate

Abstract

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).

Conclusion: 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.

Figure 1

Chromatograms showing the synthesis of AThTP (arrow) in the high molecular mass fraction (Sephadex G-200, peak I). The incubation was carried out for 1 h under the conditions described in the Methods secion in the absence (a) and the presence (b) of 1 mM ADP. The flow rate was 0.5 ml/min.

Figure 2

Time dependence of AThTP synthesis. The experiment was carried out under standard conditions (1 mM ADP, 0.1 mM ThDP, 10 mM Mg²⁺, maleate buffer, pH 6.5), and the protein concentration was 100 μg/ml.

Figure 3

Effect of pH on AThTP synthesizing enzyme. The Mg²⁺ concentration was 10 mM, and the buffers used were as follows: (○) 50 mM acetate, pH 5.5; 50 mM maleate, pH 6.0–6.5; 50 mM Tris-maleate, pH 7.0; 50 mM Tris-HCl, pH 7.5; (■) 50 mM Bis-Tris-propane; (□) 100 mM Bis-Tris-propane.

Figure 4

Effect of ThDP concentration on the activity of AThTP-synthesizing enzyme at fixed concentrations of ADP (1 mM) and Mg²⁺(10 mM). The continuous line was obtained by nonlinear regression using the Michaelis-Menten equation with an apparent K_m of 4.2 ± 0.5 mM. Inset shows the Hanes plot of the data and the line was obtained by linear regression. The results are expressed as mean ± SD for 3 experiments.

Figure 5

Effect of varying ADP concentrations on the activity of AThTP-synthesizing enzyme at a fixed ThDP concentration of 0.1 mM and 10 mM Mg²⁺. The results are the mean ± SD for 3 to 6 experiments. The line was obtained by nonlinear regression assuming a sigmoid dose-response curve with an EC₅₀ of 0.08 mM. The inset shows a Hill plot obtained for ADP concentrations ranging from 0.04 to 0.6 mM. The Hill coefficient (n_H = 2.1) was calculated from the slope of the regression line over the linear portion of the graph (0.01 – 0.20 mM ADP).

Figure 6

Effect of Mg²⁺ concentration on the activity of AThTP-synthesizing enzyme at a fixed ThDP concentration of 0.1 mM and 1 mM ADP. Total Mg²⁺ concentration (○); free Mg²⁺ concentration (●).