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 frames termed acs1 to acs4, Acs1 was 96% identical to H. influenzae type b Orf1, 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 homology to other pyrophosphorylases was only detected in the N-terminal part of Acs1, whereas the C-terminal part was homologous to several short-chain dehydrogenases/reductases, suggesting that Acs1 might be a bifunctional enzyme, 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 activities, whereas these activities were not detectable in control cells. Acs1 was purified to near homogeneity and found to copurify with ribitol 5-phosphate 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 and NADPH in one direction and for ribitol 5-phosphate and NADP(+) in the other direction and was markedly stimulated by CTP, The pyrophosphorylase showed activity with CTP and ribitol 5-phosphate or arabitol 5-phosphate, We conclude that acs1 encodes a bifunctional enzyme that converts ribulose 5-phosphate 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.