acs1 of Haemophilus influenzae type a capsulation locus region II encodes a bifunctional ribulose 5-phosphate reductase-CDP-ribitol pyrophosphorylase

The serotype-specific, 5,9-kb region II of the Haemophilus influenzae type a capsulation locus was sequenced and found to contain four open reading fr ames termed acs1 to acs4, Acs1 was 96% identical to H. influenzae type b Or f1, previously shown to have CDP-ribitol pyrophosphorylase activity (J. Van Eldere, L. Brophy, B. Loynds, P. Cells, I. Hancock, S. Carman, J. S. Kroll , and E. R. Moron, Mol. Microbiol, 15:107-118, 1995). Low but significant h omology to other pyrophosphorylases was only detected in the N-terminal par t of Acs1, whereas the C-terminal part was homologous to several short-chai n dehydrogenases/reductases, suggesting that Acs1 might be a bifunctional e nzyme, To test this hypothesis, acs1 was cloned in an expression vector and overexpressed in Escherichia coli, Cells expressing this protein displayed both ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase a ctivities, whereas these activities were not detectable in control cells. A cs1 was purified to near homogeneity and found to copurify with ribitol 5-p hosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities. These had superimposable elution profiles from DEAE-Sepharose and Blue-Sepharose columns, The dehydrogenase activity was specific for ribulose 5-phosphate a nd NADPH in one direction and for ribitol 5-phosphate and NADP(+) in the ot her direction and was markedly stimulated by CTP, The pyrophosphorylase sho wed activity with CTP and ribitol 5-phosphate or arabitol 5-phosphate, We c onclude that acs1 encodes a bifunctional enzyme that converts ribulose 5-ph osphate into ribitol 5-phosphate and further into CDP-ribitol, which is the activated precursor form for incorporation of ribitol 5-phosphate into the H. influenzae type a capsular polysaccharide.