Characterization of an NO-Activated Homodimeric Soluble Guanylate Cyclase from a Choanoflagellate

Soluble guanylate cyclases are gas-sensing enzymes in eukaryotes that catalyze the formation of cyclic GMP from GTP. While commonly studied sGCs from insects and vertebrates are heterodimers, there are additional classes of homodimeric gas-sensing sGCs in eukaryotes that are not as well characterized. Reported here is the characterization of <i>Cf</i> sGC1 isolated from the organism <i>Choanoeca flexa</i>, a single-celled eukaryote. <i>Cf</i> sGC1 is a homodimeric sGC that exhibits a three-state activation profile in response to NO similar to that observed with heterodimeric NO-responsive sGCs. <i>Cf</i> sGC1 was isolated as an active homodimer, has one heme cofactor per dimer, and exhibits typical substrate saturation kinetics. Small-angle X-ray scattering revealed that <i>Cf</i> sGC1 undergoes a structural change mirroring that of heterodimeric sGCs in the presence of excess NO (relative to the heme concentration). Additionally, two different variants of the C-terminal catalytic domain of <i>Cf</i> sGC1 (sGC1-CAT) were expressed and characterized. The <i>K</i>_d for sGC1-CAT dimerization was 1.8 ± 0.4 μM, compared to the estimated nanomolar affinity of the full-length <i>Cf</i> sGC1 construct. This implies that the N-terminal domains influence enzyme dimerization. Additionally, removal of a predicted disordered C-terminal region of an sGC1-CAT construct gave rise to a construct with ∼3x higher GTP <i>K</i>_M compared to the unmodified variant, implying that the disordered tail may enhance enzyme-substrate interactions.