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. Celis, I. Hancock, S. Carman, J. S. Kroll, and E. R. Moxon, 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.
Follens
A
A
Rega Institute for Medical Research, Catholic University of Leuven, B-3000 Leuven, B-1200 Brussels, Belgium.
Veiga-da-Cunha
M
M
Merckx
R
R
van Schaftingen
E
E
van Eldere
J
J
D000595:Amino Acid Sequence; D016667:Bacterial Capsules / Q000235:genetics*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012274:Ribulosephosphates; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
C118372:ACS1 protein, Haemophilus influenzae; D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D012274:Ribulosephosphates; C031524:ribulose 5-phosphate; D010088:Oxidoreductases; D009713:Nucleotidyltransferases; C032634:D-ribitol-5-phosphate cytidylyltransferase