CDP-6-DEOXY-DELTA(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 lipopolysacc haride of gram-negative bacteria, are essential to serological specifi city 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 enzyma tic steps, with CDP-6-deoxy-DELTA3,4-glucoseen reductase (E3) particip ating as the key enzyme in this catalysis. This enzyme has been previo usly purified from Yersinia pseudotuberculosis by an unusual procedure (protocol 1) including a trypsin digestion step (O. Han, V. P. Miller , and H.-W. Liu, J. Biol. Chem. 265:8033-8041, 1990). However, the clo ned 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 protoco l I may have been misidentified as E3. A reinvestigation of the purifi cation protocol produced a new and improved procedure (protocol 11) co nsisting of DEAE-Sephacel, phenyl-Sepharose, Cibacron blue A, and Seph adex 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 8,00 0-fold higher than that of cell lysates, and more importantly, the cor responding gene (ascD) was found to be part of the ascarylose biosynth etic cluster. Presented are the identification and confirmation of the E3 gene through cloning and overexpression and the culminating purifi cation and unambiguous assignment of homogeneous E3. The nucleotide an d translated amino acid sequences of the genuine E3 are also presented .