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 presem in the virulence-associated polysaccharide capsules of Haemophilus influenzae type a and b (Follens et al., 1999, J Bacterial. 181: 2001). In the work described here, we investigated the order of the reactions catalyzed by CDPribitol 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 ofo-ribulose 5-phosphate followed by cytidylyltransfer to oribitol 5-phosphate. Steady state kinetic measurements revealed a 650-fold kinetic preference for cytidylyltransfer too-ribitol 5-phosphate over o-ribulose 5-phosphate. Rapid mixing studies indicated quick reduction of o-ribulose 5-phosphate with a lag in the cytidylyltransfer reaction consistent with a requirement for the accumulation of Km quantities of o-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 cytidylyltransfer, respectively). Release and free diffusion of the metabolic intermediate o-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 n-ribulose 5-phosphate, release and free diffusion of the metabolic intermeditate n-ribitol 5-phosphate followed by cytidylyltransfer. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23607 |
| Date | 02 1900 |
| Creators | Zolli, Michela |
| Contributors | Brown, Dr. Eric D., Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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