Characterization of a hypotaurocyamine kinase from the protozoan, Phytophthora sojae, and its implications on the evolution of substrate specificity in the phosphagen kinase family

Abstract

Phosphagen kinases (PK) are a family of enzymes that catalyze the reversible transfer of a high‐energy gamma phosphoryl group from ATP to a guanidino acceptor molecule. A novel hypotaurocyamine kinase (HTK) from the protozoan oomycete Phytophthora sojae (PsPK) was studied to determine substrate specificity, oligomerization state, and cooperativity. Isothermal titration calorimetry was used to determine the kinetic constants of PsPK reacting with taurocyamine and, less strongly, glycocyamine by titrating the guanidino substrate at varying concentrations into a solution containing the HTK and MgATP. The Km for taurocyamine and MgATP was found to be 0.59 mM and 3.78 mM, respectively, with a kcat of 34.66 s−1, while the Km for glycocyamine and MgATP was found to be 5.1 mM and 5.1 mM, respectively, with a kcat of 3.2 s−1. Size exclusion chromatography indicates PsPK is an oligomer and isothermal titration calorimetry suggests no cooperativity among subunits. Our results coupled with findings in Monosiga suggest oligomerization evolved first in the protozoa and that cooperativity sometime later. The data also indicate there is diverse substrate specificity in protozoan PKs previously thought to exist only in annelida. This project was made possible by the support of USDA grant (USDA 2011‐68004‐30104) and the Howard Hughes Medical Institute Undergraduate Science Education Program.