MECHANISTIC STUDIES OF THE BIOSYNTHESIS OF PARATOSE - PURIFICATION AND CHARACTERIZATION OF CDP-PARATOSE SYNTHASE

The 3,6-dideoxyhexoses can be found in the cell wall lipopolysaccharid e of Gram-negative bacteria, where they have been shown to be the domi nant antigenic determinants. All naturally occurring 3,6-dideoxyhexose s, with colitose as the only exception, are biosynthesized via a compl ex pathway that begins with CDP-D-glucose. Included in this pathway is CDP-paratose synthase, an essential enzyme in the formation of the 3, 6-dideoxy sugars, CDP-paratose and CDP-tyvelose. Recently, the gene en coding CDP-paratose synthase in Salmonella typhi, rfbS, has been ident ified and sequenced [Verma, N., and Reeves, P. (1989) J. Bacteriol. 17 1, 5694-5701]. On the basis of this information, we have amplified the rfbS gene by polymerase chain reaction (PCR) from S. typhi and cloned this gene into a pET-24(+) vector. Expression and purification of CDP -paratose synthase have allowed us to fully characterize the catalytic properties of this enzyme, which is a homodimeric protein with a pref erence for NADPH over NADH. It catalyzes the stereospecific hydride tr ansfer of the pro-S hydrogen from the C-4' position of the reduced coe nzyme to C-4 of the substrate, CDP-3,6-dideoxy-D-glycero-D-glycero-4-h exulose. The overall equilibrium of this catalysis greatly favors the formation of the reduced sugar product and the oxidized coenzyme. Inte restingly, this enzyme also exhibits a high affinity for NADPH with a much smaller dissociation constant (K-ia) of 0.005 +/- 0.002 mu M comp ared to the K-m of 26 +/- 8 mu M for NADPH. While this unusual propert y complicated the interpretation of the kinetic data, the kinetic mech anism of CDP-paratose synthase as explored by the combination of bisub strate kinetic analysis, product inhibition studies, and dead-end comp etitive inhibition studies is most consistent with a Theorell-Chance m echanism. The present study on CDP-paratose synthase, a likely new mem ber of the short-chain dehydrogenase family, represents the first deta iled characterization of this type of ketohexose reductase, many of wh ich may share similar properties with CDP-paratose synthase.