The enzymatic synthesis of cytidine monophospho-2-keto-3-deoxy-octonate.

The accompanying paper (Heath and Ghalambor, 1963) presents evidence for the occurrence of 2-keto-3-deoxy-octonate @DO) as a glycosidically bound constituent in preparations of cell wall lipopolysaccharide (LPS) obtained from Escherichia cs 0111-B4. The purpose of this paper is to report the biosynthesis of cytidine monophospho-RDO (CMP-KDO) by an enzyme purified from this organism. Materials and Methods--Unless otherwise noted, all compounds, reagents, and methods used in these studies were the same as those indicated in the preceding paper; abbreviations used here are also the same. Enzyme purification--The assay procedure is based on the principle that 2-keto-3-deoxy-onic acids no longer give a positive reaction in the TBA method after the carbonyl group is reduced with NaBH& (Weissbach and liurwitx, 1959). Thus, after incubation of CTP, RDO and enzyme, the remaining uncombined RDO is reduced with NaBH4 while the CMp-bound KDO is resistant to reduction. After destruction of the excess borohydride with acetone, followed by mild acid hydrolysis, the KDO released from CMP-RDO was measured by the TBA mathod. Using this assay procedure, the formation of CMP-ND0 was linear for 30 min, over a 5-fold range in protein concentration.