Publications

• Identification and sequence of rfbS and rfbE, which determine antigenic specificity of group A and group D salmonellae.

  Verma N., Reeves P.R.

  Salmonella group A, group B, and group D strains have paratose, abequose, and tyvelose, respectively, as the immunodominant sugar in their O antigens, which are otherwise identical; only the final steps differ in the biosynthetic pathways of these sugars. The gene rfbJ from a group B strain, encod ... >> More

  Salmonella group A, group B, and group D strains have paratose, abequose, and tyvelose, respectively, as the immunodominant sugar in their O antigens, which are otherwise identical; only the final steps differ in the biosynthetic pathways of these sugars. The gene rfbJ from a group B strain, encoding abequose synthase, the final and only unique step in the biosynthesis of CDP-abequose, has been cloned and sequenced (P. Wyk and P. Reeves, J. Bacteriol. 171:5687-5693, 1989). In this study, we locate and sequence rfbS and rfbE from serovars typhi and paratyphi, representative of groups A and D. Gene rfbS is present in both groups and encodes paratose synthase, which carries out a step parallel to that of abequose synthase, but the product is CDP-paratose. The DNA and inferred amino acid sequences are compared with those of rfbJ. We conclude that the genes are homologous, but the divergence is extremely ancient. Gene rfbE encodes CDP-tyvelose epimerase, which converts CDP-paratose to CDP-tyvelose in group D strains; the gene is active in group D strains, and we find it to be present in a mutant form in group A strains. These two genes encode the steps unique to groups A and D and, like rfbJ of group B, are of low G+C content, suggesting transfer from outside of salmonellae. The evolutionary origin of these genes is discussed. << Less

  J. Bacteriol. 171:5694-5701(1989) [PubMed] [EuropePMC]

• Mechanistic studies of the biosynthesis of paratose: purification and characterization of CDP-paratose synthase.

  Hallis T.M., Lei Y., Que N.L., Liu H.

  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 be ... >> More

  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. << Less

  Biochemistry 37:4935-4945(1998) [PubMed] [EuropePMC]