Regulation of prokaryotic adenylyl cyclases by CO2.

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.

The effect of HAMP domains on class IIIb adenylyl cyclases from Mycobacterium tuberculosis.

The genes Rv1318c, Rv1319c, Rv1320c and Rv3645 of Mycobacterium tuberculosis are predicted to code for four out of 15 adenylyl cyclases in this pathogen. The proteins consist of a membrane anchor, a HAMP region and a class IIIb adenylyl cyclase catalytic domain. Expression and purification of the isolated catalytic domains yielded adenylyl cyclase activity for all four recombinant proteins. Expression of the HAMP region fused to the catalytic domain increased activity in Rv3645 21-fold and slightly reduced activity in Rv1319c by 70%, demonstrating isoform-specific effects of the HAMP domains. Point mutations were generated to remove predicted hydrophobic protein surfaces in the HAMP domains. The mutations further stimulated activity in Rv3645 eight-fold, whereas the effect on Rv1319c was marginal. Thus HAMP domains can act directly as modulators of adenylyl cyclase activity. The modulatory properties of the HAMP domains were confirmed by swapping them between Rv1319c and Rv3645. The data indicate that in the mycobacterial adenylyl cyclases the HAMP domains do not display a uniform regulatory input but instead each form a distinct signaling unit with its adjoining catalytic domain.

A defined subset of adenylyl cyclases is regulated by bicarbonate ion.

The molecular basis by which organisms detect and respond to fluctuations in inorganic carbon is not known. The cyaB1 gene of the cyanobacterium Anabaena sp. PCC7120 codes for a multidomain protein with a C-terminal class III adenylyl cyclase catalyst that was specifically stimulated by bicarbonate ion (EC50 9.6 mm). Bicarbonate lowered substrate affinity but increased reaction velocity. A point mutation in the active site (Lys-646) reduced activity by 95% and was refractory to bicarbonate activation. We propose that Lys-646 specifically coordinates bicarbonate in the active site in conjunction with an aspartate to threonine polymorphism (Thr-721) conserved in class III adenylyl cyclases from diverse eukaryotes and prokaryotes. Using recombinant proteins we demonstrated that adenylyl cyclases that contain the active site threonine (cyaB of Stigmatella aurantiaca and Rv1319c of Mycobacterium tuberculosis) are bicarbonate-responsive, whereas adenylyl cyclases with a corresponding aspartate (Rv1264 of Mycobacterium) are bicarbonate-insensitive. Large numbers of class III adenylyl cyclases may therefore be activated by bicarbonate. This represents a novel mechanism by which diverse organisms can detect bicarbonate ion.