title: Mechanistic studies of the biosynthesis of paratose: purification and characterization of CDP-paratose synthase.
abstract: The 3,6-dideoxyhexoses can be found in the cell wall lipopolysaccharide of Gram-negative bacteria, where they have been shown to be the dominant antigenic determinants. All naturally occurring 3,6-dideoxyhexoses, with colitose as the only exception, are biosynthesized via a complex 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 encoding CDP-paratose synthase in Salmonella typhi, rfbS, has been identified and sequenced [Verma, N., and Reeves, P. (1989) J. Bacteriol. 171, 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 preference for NADPH over NADH. It catalyzes the stereospecific hydride transfer of the pro-S hydrogen from the C-4' position of the reduced coenzyme to C-4 of the substrate, CDP-3,6-dideoxy-D-glycero-D-glycero-4-hexulose. The overall equilibrium of this catalysis greatly favors the formation of the reduced sugar product and the oxidized coenzyme. Interestingly, this enzyme also exhibits a high affinity for NADPH with a much smaller dissociation constant (Kia) of 0.005 +/- 0.002 microM compared to the Km of 26 +/- 8 microM for NADPH. While this unusual property complicated the interpretation of the kinetic data, the kinetic mechanism of CDP-paratose synthase as explored by the combination of bisubstrate kinetic analysis, product inhibition studies, and dead-end competitive inhibition studies is most consistent with a Theorell-Chance mechanism. The present study on CDP-paratose synthase, a likely new member of the short-chain dehydrogenase family, represents the first detailed characterization of this type of ketohexose reductase, many of which may share similar properties with CDP-paratose synthase.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: T M
authors.initials: TM
authors.lastname: Hallis
authors.forename: Y
authors.initials: Y
authors.lastname: Lei
authors.forename: N L
authors.initials: NL
authors.lastname: Que
authors.forename: H
authors.initials: H
authors.lastname: Liu
mesh_terms: D002238:Carbohydrate Epimerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006601:Hexoses / Q000096:biosynthesis*; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D006601:Hexoses; D011994:Recombinant Proteins; D009249:NADP; C022880:paratose; D002238:Carbohydrate Epimerases; C061271:paratose synthase