ribulose-5-phosphate and cytidine-diphosphate-ribitol

The cell wall of many pathogenic Gram-positive bacteria contains ribitol-phosphate wall teichoic acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-ribitol, the activated precursor for ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-ribitol synthesis from CTP and ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-ribitol production that may aid in the design of inhibitors against WTA biosynthesis.

Topics: Bacterial Proteins; Catalytic Domain; Cell Wall; Crystallography, X-Ray; Mass Spectrometry; Models, Molecular; Mutation; Nucleoside Diphosphate Sugars; Nucleotidyltransferases; Oxidoreductases; Pentosephosphates; Protein Multimerization; Ribulosephosphates; Staphylococcus aureus; Teichoic Acids

CDP-ribitol synthase catalyzes the formation of CDP-ribitol from ribulose 5-phosphate, NADPH, and CTP. CDP-ribitol is an activated precursor for the synthesis of virulence-associated polysaccharides in the capsule of the Gram-negative pathogen Haemophilus influenzae and in the cell walls of Gram-positive pathogens including Staphylococcus aureus. We showed previously that CDP-ribitol synthase activity in H. influenzae is catalyzed by the bifunctional enzyme Bcs1 in a two-step reaction with reduction preceding cytidylyl transfer [Zolli, M., et al. (2001) Biochemistry 40, 5041-5048]. In the work reported here, we predicted a CDP-ribitol synthesis locus in S. aureus tandemly arranged as tarI, encoding an orthologue of the cytidylyltransferase domain of Bcs1, and tarJ, coding for an analogue of the reductase domain of Bcs1. We have shown the formation of a functional CDP-ribitol synthase complex between TarI and TarJ. Steady-state mechanistic studies of the CDP-ribitol synthases TarIJ and Bcs1 revealed that the analogous reductases and orthologous cytidylyltransferases undergo ordered mechanisms. The sequence of substrate binding and product release of the orthologous cytidylyltransferases differed. Steady-state analysis of the reductase and cytidylyltransferase activities of TarIJ indicated a 100-fold difference in the turnover where the primary reductase was rate limiting. Rapid mixing experiments revealed the presence of approximately 12 microM ribitol 5-phosphate at steady state, 100-fold lower than the observed K(m) for this intermediate. Analysis of the approach to steady state suggested that channeling was not occurring in the coupled enzyme complex and was an unlikely driving force in the convergent recruitment of reductase and cytidylyltransferase activities in the two CDP-ribitol synthases.

Topics: Bacterial Proteins; Binding Sites; Haemophilus influenzae; Molecular Structure; Molecular Weight; NADP; Nucleoside Diphosphate Sugars; Nucleotidyltransferases; Oxidoreductases; Pentosephosphates; Protein Binding; Ribulosephosphates; Staphylococcus aureus

CDP-ribitol synthase is a bifunctional reductase and cytidylyltransferase that catalyzes the transformation of D-ribulose 5-phosphate, NADPH, and CTP to CDP-ribitol, a repeating unit present in the virulence-associated polysaccharide capsules of Haemophilus influenzae types a and b [Follens, A., et al. (1999) J. Bacteriol. 181, 2001]. In the work described here, we investigated the order of the reactions catalyzed by CDP-ribitol synthase and conducted experiments to resolve the question of substrate channeling in this bifunctional enzyme. It was determined that the synthase first catalyzed the reduction of D-ribulose 5-phosphate followed by cytidylyl transfer to D-ribitol 5-phosphate. Steady state kinetic measurements revealed a 650-fold kinetic preference for cytidylyl transfer to D-ribitol 5-phosphate over D-ribulose 5-phosphate. Rapid mixing studies indicated quick reduction of D-ribulose 5-phosphate with a lag in the cytidylyl transfer reaction, consistent with a requirement for the accumulation of K(m) quantities of D-ribitol 5-phosphate. Signature motifs in the C-terminal and N-terminal sequences of the enzyme (short chain dehydrogenase/reductase and nucleotidyltransferase motifs, respectively) were targeted with site-directed mutagenesis to generate variants that were impaired for only one of the two activities (K386A and R18A impaired for reduction and cytidylyl transfer, respectively). Release and free diffusion of the metabolic intermediate D-ribitol 5-phosphate was indicated by the finding that equimolar mixtures of K386A and R18A variants were efficient for bifunctional catalysis. Taken together, these findings suggest that bifunctional turnover occurs in distinct active sites of CDP-ribitol synthase with reduction of D-ribulose 5-phosphate and release and free diffusion of the metabolic intermediate D-ribitol 5-phosphate followed by cytidylyl transfer.

Topics: Binding Sites; Catalysis; Haemophilus influenzae; Kinetics; Multienzyme Complexes; Mutagenesis, Site-Directed; Nucleoside Diphosphate Sugars; Nucleotidyltransferases; Oxidation-Reduction; Pentosephosphates; Recombinant Proteins; Ribulosephosphates; Substrate Specificity