CDP-6-deoxy-Δ^3,4-glucoseen reductase from Yersinia pseudotuberculosis: Enzyme purification and characterization of the cloned gene

The 3,6-dideoxyhexoses, usually confined to the cell wall lipopolysaccharide of gram-negative bacteria, are essential to serological specificity and are formed via a complex biosynthetic pathway beginning with CDP-D-hexoses. In particular, the biosynthesis of CDP-ascarylose, one of the naturally occurring 3,6-dideoxyhexoses, consists of five enzymatic steps, with CDP-6-deoxy-Δ^3,4-glucoseen reductase (E₃) participating as the key enzyme in this catalysis. This enzyme has been previously purified from Yersinia pseudotuberculosis by an unusual procedure (protocol I) including a trypsin digestion step (O. Han, V. P. Miller, and H.-W. Liu, J. Biol. Chem. 265:8033-8041, 1990). However, the cloned gene showed disparity with the expected gene characteristics, and upon expression, the resulting gene product exhibited no E₃ activity. These findings strongly suggested that the protein isolated by protocol I may have been misidentified as E₃. A reinvestigation of the purification protocol produced a new and improved procedure (protocol II) consisting of DEAE-Sephacel, phenyl-Sepharose, Cibacron blue A, and Sephadex G-100 chromatography, which efficiently yielded a new homogeneous enzyme composed of a single polypeptide with a molecular weight of 39,000. This highly purified protein had a specific activity nearly 8,000-fold higher than that of cell lysates, and more importantly, the corresponding gene (ascI) was found to be part of the ascarylose biosynthetic cluster. Presented are the identification and confirmation of the E₃ gene through cloning and overexpression and the culminating purification and unambiguous assignment of homogeneous E₃. The nucleotide and translated amino acid sequences of the genuine E₃ are also presented.