Cystathionine β-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria

Abstract

Among numerous proteins containing pairs of regulatory cystathionine β-synthase (CBS) domains, family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP or ADP) or activation (ATP). Here we show that linear P(1),P(n)-diadenosine 5'-polyphosphates (ApnAs, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi, and Clostridium perfringens and increase their activity up to 30-, 5-, and 7-fold, respectively. Ap4A, Ap5A, and Ap6A bound noncooperatively and with similarly high affinities to CBS-PPases, whereas Ap3A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All ApnAs abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were ~10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap4A and Ap5A but 5.5 kcal/mol and 2 mol/mol for Ap3A, AMP, ADP, and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain no DRTGG domain, were not affected by ApnAs and showed no enthalpy change, indicating the importance of the DTRGG domain for ApnA binding. These findings suggest that ApnAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria.

Keywords: CBS domain; allosteric regulation; bacterial signal transduction; calorimetry; cooperativity; diadenosine polyphosphate; enzyme kinetics; inorganic pyrophosphatase; stress response.

FIGURE 1.

Concentration dependences of the effects of Ap_nAs on the activity of three CBS-PPases measured at fixed concentrations of substrate (50 μm MgPP_i) and Mg²⁺ (5 mm). The lines show the best fits of Equations 1 or 4 (see text for details). Activity without nucleotides (220, 350, and 800 s⁻¹ for dhPPase, cnPPase and cpPPase, respectively) was taken as unity. dh, dhPPase; cn, cnPPase; cp, cpPPase.

FIGURE 2.

Mg²⁺ concentration dependence of CBS-PPase activation by Ap₃A (left panel) and Ap₄A (right panel). The panels show (from top to bottom) the activation factor K_N1 (○) and K_N2 (●) values and Hill coefficients. The K_N1 and K_N2 lines show the best fits to Equation 3. The horizontal dotted lines (h = 1) mark the boundary between positive and negative cooperativity. dh, dhPPase; cn, cnPPase; cp, cpPPase.

FIGURE 3.

Lack of kinetic cooperativity in CBS-PPases in the presence of 10 μm Ap₄A. The panels show (from top to bottom) the catalytic constant k_cat, the Michaelis constant K_m, and the Hill coefficient h. The dashed lines and corresponding points refer to the early estimated parameter values in the absence of Ap₄A (32); K_m values refer to the average Michaelis constants ($\sqrt{K_{\text{m}1}{K}_{\text{m}2}}$) in this case. The horizontal dotted lines (h = 1) mark the boundary between positive and negative cooperativity. K_m values are measured in terms of the MgPP_i complex. dh, dhPPase; cn, cnPPase; cp, cpPPase.

FIGURE 4.

Mg²⁺ concentration dependence of k_cat for cpPPase measured in the presence of 50 μm Ap₃A or 10 μm Ap₄A or Ap₅A. The values of k_cat were fit to the equation k_cat = k_cat,0 + (k_cat,M − k_cat,0)/{1 + (K_m/[M])²}, where k_cat,0 and k_cat,M are the limiting values of k_cat at 0 and infinite Mg²⁺ concentrations, respectively, and K_m is the metal binding constant.

FIGURE 5.

Ap₄A concentration dependence of kinetic cooperativity in cpPPase in the presence of 0.5 mm Mg²⁺. Notations are as in Fig. 4. The values of k_cat were fit to the equation k_cat = k_cat,0 + (k_cat,N − k_cat,0)/(1 + K_N/[M]), where k_cat,0 and k_cat,M are the limiting values of k_cat at 0 and infinite Ap₄A concentrations, respectively, and K_N is the nucleotide binding constant. The line for K_m1 shows the best fit to Equation 3.

FIGURE 6.

ITC measurements of nucleotide binding to CBS-PPases. A, typical raw data for successive injections of Ap₄A into dhPPase solution. B, integrated heats for titration of four CBS-PPases by selected mono- and dinucleotides after correction for dilution. Enzyme dimer concentration was 4 μm (dhPPase), 5 μm (cnPPase), 3.5 μm (cpPPase), or 2.5 μm (elPPase). The lines show the best fits for a single-binding site model.

FIGURE 7.

Aligned amino acid sequences of the two CBS domains of the characterized CBS-PPases. Amino acid residues making contacts with Ap₄A or AMP in the crystal structures of cpPPase (33) are shown in boxes. Consensus residues based on 180 CBS-PPase sequences are indicated in the two bottom lines. Residue numbering is for full-length cpPPase. Consensus residues for different levels of identity are indicated below the set of sequences.