Unique evolution of Bivalvia arginine kinases

Abstract

The clams Pseudocardium, Solen, Corbicula and Ensis possess a unique form of arginine kinase (AK) with a molecular mass of 80 kDa and an unusual two-domain structure, a result of gene duplication and subsequent fusion. These AKs also lack two functionally important amino acid residues, Asp⁶² and Arg¹⁹³, which are strictly conserved in other 40-kDa AKs and are assumed to be key residues for stabilizing the substrate-bound structure. However, these AKs show higher enzyme activity. The cDNA-derived amino acid sequences of 40-kDa AKs from the blood clam Scapharca broughtonii and the oyster Crassostrea gigas were determined. While Asp⁶² and Arg¹⁹³ are conserved in Scapharca AK, these two key residues are replaced by Asn and Lys, respectively, in Crassostrea AK. The native enzyme from Crassostrea and both of the recombinant enzymes show an enzyme activity similar to that of two-domain clam AKs and at least twofold higher than that of other molluskan AKs. Although the replacement of Asp⁶² or Arg¹⁹³ by Gly in normal AK causes a considerable decrease in V_max (6–15% of wild-type enzyme) and a two- to threefold increase in K_m for arginine, the same replacement in Scapharca AK had no pronounced effect on enzyme activity. Together with the observation that bivalve AKs are phylogenetically distinct from other molluskan AKs, these results suggest that bivalve AKs have undergone a unique molecular evolution; the characteristic stabilizing function of residues 62 and 193 has been lost and, consequently, the enzyme shows higher activity than normal.