Biosynthesis of KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) - Identification and characterization of a KDN-9-phosphate synthetase activity from trout testis

Although the deaminoneuraminic acid or KDN glycotope (2-keto-3-deoxy-D-glyc ero-D-galacto-nononic acid) is expressed in glycoconjugates that range in e volutionary diversity from bacteria to man, there is little information as to how this novel sugar is synthesized. Accordingly, biosynthetic studies w ere initiated in trout testis, an organ rich in KDN, to determine how this sialic acid is formed. These studies have shown that the pathway consists o f the following three sequential reactions: 1) Man + ATP --> Man-6-P + ADP; 2) Man-6-P + PEP --> KDN-9-P + Pi; 3) KDN-9-P --> KDN + Pi. Reaction 1, ca talyzed by a hexokinase, is the 6-O-phosphorylation of mannose to form D-ma nnose 6-phosphate (Man-6-P). Reaction 2, catalyzed by KDN-9-phosphate (KDN- 9-P) synthetase, condenses Man-6-P and phosphoenolpyruvate (PEP) to form KD N-9-P. Reaction 3, catalyzed by a phosphatase, is the dephosphorylation of KDN-9-P to yield free KDN. It is not known if a kinase specific for Man (Re action 1) and a phosphatase specific for KDN9-P (Reaction 3) may exist in t issues actively synthesizing KDN. In this study, the KDN-9-P synthetase, an enzyme that has not been previously described, was identified as at least one key enzyme that is specific for the KDN biosynthetic pathway. This enzy me was purified 50-fold from rainbow trout testis and characterized. The mo lecular weight of the enzyme was estimated to be about 80,000, and activity was maximum at neutral pH in the presence of Mn2+. N-Acetylneuraminic acid g-phosphate (Neu5Ac-9-P) synthetase, which catalyzes the condensation of N -acetyl-D-mannosamine 6-phosphate and phosphoenol-pyruvate to produce Neu5A c-9-P, was co-purified with the KDN-9-P synthetase. Substrate competition e xperiments revealed, however, that syntheses of KDN-9-P and Neu5Ac-9-P were catalyzed by two separate synthetase activities. The significance of these studies takes on added importance with the recent discovery that the level of free KDN is elevated in human fetal cord but not matched adult red bloo d cells and in ovarian cancer cells (Inoue, S., Lin, S-L., Chang, T., Wu, S -H., Yao, C-W., Chu, T-Y., Troy, F. A., II, and Inoue, Y. (1998) J. Biol. C hem. 273, 27199-27204). This unexpected finding emphasizes the need to unde rstand more fully the role that free KDN and KDN-glycoconjugates may play i n normal hematopoiesis and malignancy.