A novel arginine kinase from the shrimp Neocaridina denticulata: the fourth arginine kinase gene lineage.

Arginine kinases (AK) evolved independently at least three times: first at an early stage of phosphagen kinase evolution (typical AK), second from the cytoplasmic creatine kinase (CK) gene later in metazoan evolution (Stichopus AK) and third from the mitochondrial CK gene in the course of annelid radiation (Sabellastarte AK). Here, we present a possible fourth AK lineage. We amplified cDNA encoding three AKs (AK1, AK2 and AK3) from the shrimp Neocaridina denticulata, and determined their amino acid sequences (355-356 residues). Each cDNA sequence was cloned in a pET plasmid and the corresponding recombinant kinase was expressed in E. coli. The AKs showed monomeric nature similar to typical AKs on an analytical gel filtration column. While the amino acid sequence of AK2 corresponded to that of typical AK, containing the conserved key residues established in Limulus AK for the substrate binding site, those of AK1 and AK3 lacked some of these key residues, indicating a similar evolution to Stichopus and Sabellastarte AKs. Moreover, phylogenetic analysis of phosphagen kinases indicated that Neocaridina AK1 and AK3 diverged at the deepest branching point close to the root of the tree and formed a distinct cluster outside the typical AK cluster, which included Neocaridina AK2. Kinetic constants of Neocaridina AKs were similar to those of other AKs. However, activation energy (E(a)) for the transition state of AK1 and AK3 was about 1.5-fold larger than that of AK2. The DeltaH(o++) values for AK1 and AK3 were also about 1.5-fold larger than that for AK2, but all three DeltaG(o++) values were very similar (71-72 kJ/mol); this results in similar reaction velocities for the three AK reactions due to a lower decrease in entropy in the activated complexes of AK1 and AK3 reactions compared to that of AK2. These findings suggest that Neocaridina AK1 and AK3 are unique from the known three AK gene lineages and therefore comprises a fourth AK lineage.