Regulation of prokaryotic adenylyl cyclases by CO2

Abstract

The Slr1991 adenylyl cyclase of the model prokaroyte Synechocystis PCC 6803 was stimulated 2-fold at 20 mM total C(i) (inorganic carbon) at pH 7.5 through an increase in k(cat). A dose response demonstrated an EC50 of 52.7 mM total C(i) at pH 6.5. Slr1991 adenylyl cyclase was activated by CO2, but not by HCO3-. CO2 regulation of adenylyl cyclase was conserved in the CyaB1 adenylyl cyclase of Anabaena PCC 7120. These adenylyl cyclases represent the only identified signalling enzymes directly activated by CO2. The findings prompt an urgent reassessment of the activating carbon species for proposed HCO3--activated adenylyl cyclases.

Figure 1. AC activity of purified recombinant Slr1991_120–337

(a) Purification of recombinant Slr1991_120–337 (SDS/PAGE analysis and Coomassie Blue staining). A 1.5 μg portion of protein was applied and molecular-mass standards (in kDa) are indicated. (b) Slr1991_120–337 specific activity (n=8) in the presence of 20 mM total C_i/salt (0.6 μM protein and 20 μM Mn²⁺-ATP, pH 7.5).

Figure 2. Response of wild-type Slr1991_120–337 to C_i

Slr1991_120–337 specific activity (n=6) was plotted against increasing total C_i (‘inorganic carbon’). The assay mixture contained 1.5 μM protein and 20 μM Mn²⁺-ATP, pH 6.5, with Na⁺ as cation. The total salt concentration was adjusted to 200 mM with NaCl.

Figure 3. Activation of AC by CO₂

(a) Ratio of the specific activities of Slr1991_120–337 when assayed in the presence of 40 mM total C_i or NaCl at various pH values (8 μM protein, 1 mM Mg²⁺-ATP and 20 mM Mg²⁺). The inset shows the percentage of total C_i made up by CO₂ and HCO₃⁻ over the pH range tested. (b) Change in pH of a 5 mM Mes solution (starting pH 6.4) on addition of 20 mM NaHCO₃ (↑) in the presence (□) or absence (△) of 132 units of carbonic anhydrase at 0 °C. (c) cAMP produced by Slr1991_120–337 under conditions of C_i disequilibrium (50 μM Slr1991_120–337 protein, 0 °C, 10 s, 20 mM CO₂/NaHCO₃/NaCl, 100 mM Mes, pH 6.5, 150 μM Mn²⁺-ATP). (d), cAMP produced by CyaB1_595–859 under conditions of C_i disequilibrium (38 μM CyaB1_595–859 protein, 0 °C, 10 s, 20 mM CO₂/NaHCO₃/NaCl, 100 mM Mes, pH 6.5, 150 μM Mn²⁺-ATP, n=6).