Molecular cloning of a unique CMP-sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates

2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a sialic acid (Sia ) that is ubiquitously expressed in vertebrates during normal development a nd tumorigenesis. Its expression is thought to be regulated by multiple bio synthetic steps catalyzed by several enzymes, including CMP-Sia synthetase. Using crude enzyme preparations, it was shown that mammalian CMP-Sia synth etases had very low activity to synthesize CMP-KDN from KDN and CTP, and th e corresponding enzyme from rainbow trout testis had high activity to synth esize both CMP-KDN and CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al [ 1993] J. Biol Chem., 268, 2640-2648). To demonstrate if the unique substrat e specificity found in the crude trout enzyme is conveyed by a single enzym e, cDNA cloning of trout CMP-Sia synthetase was carried out by PCR-based st rategy. The trout enzyme was shown to consist of 432 amino acids with two p otential nuclear localization signals, and the cDNA sequence displayed 53.8 % identity to that of the murine enzyme. Based on the V-max/K-m values, the recombinant trout enzyme had high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min(-1)). In contrast, the recombinant murine enzyme had 15 ti mes lower activity toward KDN than Neu5Ac (0.23 versus 3.5 min(-1)). Northe rn blot analysis suggested that several sizes of the mRNA are expressed in testis, ovary, and liver in a tissue-specific manner. These results indicat e that at least one cloned enzyme has the ability to utilize both KDN and N eu5Ac as substrates efficiently and is useful for the production of CMP-KDN .