Cloning and expression of a membrane-bound CMP-N-acetyineuraminic acid hydroxylase from the starfish Asterias rubens

The sialic acid N-glycolylneuraminic acid (Neu5Gc) is synthesized by the ac tion of CMP-Neu5Ac hydroxylase. The enzyme from various mammals has been pu rified, characterized and sequenced by cDNA cloning. Although functional se quence motifs can be postulated from comparisons with several enzymes, no g lobal homologies to any other proteins have been found. The unusual charact eristics of this hydroxylase raise questions about its evolution. As echino derms are phylogenetically the oldest organisms possessing Neu5Gc, they rep resent a starting point for investigations on the origin of this enzyme. De spite many similarities with its mammalian counterpart, CMP-Neu5Ac hydroxyl ase from the starfish A. rubens exhibits fundamental differences, most nota bly its association with a membrane and a requirement for high ionic streng th. In order to shed light on the structural basis for these differences, t he primary structure of CMP-Neu5Ac hydroxylase from A. rubens has been dete rmined by PCR and cDNA-cloning techniques, using initial sequence informati on from the mouse enzyme. The complete assembled cDNA contained an ORF codi ng for a protein of 653 amino acids with a molecular mass of 75 kDa. The de duced amino-acid sequence exhibited a high degree of homology with the mamm alian enzyme, although the C-terminus was some 60 residues longer. This ext ension consists of a terminal hydrophobic region, which may mediate membran e-binding, and a preceeding hydrophilic sequence which probably serves as a hinge or linker. The identity of the ORF was confirmed by expression of ac tive CMP-Neu5Ac hydroxylase in E. coli at low temperatures.