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Opheline kinase (OK) is one of the phosphagen kinases (PKs) restricted to annelids, but the amino acid sequence has not been determined yet. The OK enzyme was isolated in 1966 from the polychaete Ophelia neglecta (Opheliidae) and shown to have somewhat broader activities for the various substrates opheline, lombricine and taurocyamine. To determine the OK sequence, we analyzed the RNA sequencing data for Ophelina sp. and Thoracophelia sp., belonging to Opheliidae. Four PK sequences, namely, taurocyamine kinase (TK), creatine kinase (CK), mitochondrial CK (MiCK) and putative OK, were identified in both species, and the recombinant Ophelina enzymes were expressed in E. coli and purified. Since the substrate opheline was not commercially available, we used the partial activity toward taurocyamine to infer the enzyme specificity. The putative Ophelina OK showed lower activity to taurocyamine with a V max /K m nearly identical to a previously published value for an OK from a related species Ophelia neglecta. Under the same conditions, the true Ophelina TK showed much higher activity. Thus, the putative Ophelina enzyme was determined to be OK. The amino acid sequence alignment indicated that Ophelina and Thoracophelia OKs have five amino acid deletions in the GS region, like those of LKs and AKs, and the guanidino substrate specific residue was Lys, the same as LKs. In the phylogenetic tree constructed from annelid PK amino acid sequences, the OK sequences formed a distinct cluster, and it was placed near the TK and lombricine kinase (LK) clusters. This is the first report of the amino acid sequence for the OK enzyme., (Copyright © 2021 Elsevier Inc. All rights reserved.)