Reduction precedes cytidylyl transfer without substrate channeling in distinct active sites of the bifunctional CDP-ribitol synthase from Haemophilusinfluenzae
M. Zolli et al., Reduction precedes cytidylyl transfer without substrate channeling in distinct active sites of the bifunctional CDP-ribitol synthase from Haemophilusinfluenzae, BIOCHEM, 40(16), 2001, pp. 5041-5048
CDP-ribitol synthase is a bifunctional reductase and cytidylyltransferase t
hat catalyzes the transformation of D-ribulose 5-phosphate, NADPH, and CTP
to CDP-ribitol, a repeating unit present in the virulence-associated polysa
ccharide capsules of Haemophilus influenzae types a and b [Follens, A., et
al. (1999) J. Bacteriol. 181, 2001]. In the work described here, we investi
gated the order of the reactions catalyzed by CDP-ribitol synthase and cond
ucted experiments to resolve the question of substrate channeling in this b
ifunctional enzyme. It was determined that the synthase first catalyzed the
reduction of D-ribulose 5-phosphate followed by cytidylyl transfer to D-ri
bitol 5-phosphate. Steady state kinetic measurements revealed a 650-fold ki
netic preference for cytidylyl transfer to D-ribitol 5-phosphate over D-rib
ulose 5-phosphate. Rapid mixing studies indicated quick reduction of D-ribu
lose 5-phosphate with a lag in the cytidylyl transfer reaction, consistent
with a requirement for the accumulation of K, quantities of D-ribitol 5-pho
sphate. Signature motifs in the C-terminal and N-terminal sequences of the
enzyme (short chain dehydrogenase/reductase and nucleotidyltransferase moti
fs, respectively) were targeted with site-directed mutagenesis to generate
variants that were impaired for only one of the two activities (K386A and R
18A impaired for reduction and cytidylyl transfer, respectively). Release a
nd free diffusion of the metabolic intermediate D-ribitol 5-phosphate was i
ndicated by the finding that equimolar mixtures of K386A and R18A variants
were efficient for bifunctional catalysis. Taken together, these findings s
uggest that bifunctional turnover occurs in distinct active sites of CDP-ri
bitol synthase with reduction of D-ribulose 5-phosphate and release and fre
e diffusion of the metabolic intermeditate D-ribitol 5-phosphate followed b
y cytidylyl transfer.