The characterization of novel monomeric creatine kinases in the early branching Alveolata species, Perkinsus marinus: Implications for phosphagen kinase evolution

Abstract

The genome of the unicellular molluscan parasite Perkinsus marinus contains at least five genes coding for putative creatine kinases (CK), a phosphoryl transfer enzyme which plays a key role in cellular energy transactions. Expression and kinetic analyses of three of the P. marinus CKs revealed them to be true CKs with catalytic properties in the range of typical metazoan CKs. A sequence comparison of the P. marinus CKs with a range of CK dimers and other dimeric phosphoryl transfer enzymes in this family (phosphagen kinases) showed that the P. marinus CKs lacked some of the critical residues involved in dimer stabilization, a trait all previously characterized CKs share. Size exclusion chromatography of all three expressed P. marinus CK constructs indicated they are monomeric, consistent with the observed lack of some critical dimer stabilizing residues. Phylogenetic analyses of the P. marinus CKs and putative dinoflagellate CKs with a broad range of monomeric and dimeric phosphagen kinases revealed that the Perkinsus CKs form a distinct, well-supported clade with dinoflagellate CKs which also lack the dimer stabilizing residues. Analysis of the genomic data for P. marinus showed the presence of putative genes for the two enzymes associated with creatine biosynthesis. CK in higher organisms plays a critical role in energy buffering in cell types displaying high and variable rates of ATP turnover. The presence of multiple CKs and the creatine biosynthetic pathway in P. marinus indicates that this unicellular parasite has the full complement of molecular machinery for CK-mediated energy buffering.

Keywords: Creatine kinase; Enzyme kinetics; Perkinsozoa; Phosphagen kinases; Protein evolution; Protista; Quaternary structure.