Expression and characterization of phytophthora sojae phosphagen kinases

Allyson Kaitlin Palmer, The College of Wooster

Abstract

Phosphagen kinases (PKs) are a family of enzymes that are distributed throughout the animal kingdom as well as some protozoan and bacterial species. These enzymes play a critical role in energy homeostasis by catalyzing the reversible transfer of a high energy phosphoryl group from ATP to an acceptor molecule containing a guanidino group. Creatine kinase (CK), which is found mainly in vertebrates and some invertebrates, exists as a dimer. Arginine kinase (AK), found in invertebrates, protozoa, and some bacteria, usually exists as a monomeric enzyme, but several dimeric AKs have recently been characterized. The evolutionary relationship between these two forms of AK and the CK is unclear. Dimeric AKs described in invertebrates in the past were proposed to have derived from CK, however multiple occurrences of this "back evolution" make this scenario problematic. Using certain sequence motifs thought to be important for dimerization in PKs, two putative PK genes were identified in a protozoan, the oomycete Phytophthora sojae, that were predicted to encode dimeric enzymes. This study aimed to shed light on the evolutionary pathway of phosphagen kinases by investigating the quaternary structure and substrate specificity of these two phosphagen kinases. These putative dimeric PKs were cloned and expressed and shown to exhibit taurocyamine kinase activity, which suggests that the preferred substrate of these enzymes is taurocyamine or hypotaurocyamine. This is the first report of such substrate specificity within the protozoa. In addition, Native-PAGE and size-exclusion chromatography data indicate that they do in fact adopt dimeric quaternary structure. These data suggest that dimeric PKs evolved much earlier than previously thought and indicates that protozoan PKs may exhibit diversity in substrate specificity that was previously thought to exist only in annelida.

 

© Copyright 2010 Allyson Kaitlin Palmer