Fig. 1: Bcs3 is the capsule polymerase of Hib.

a, Chemical structure and schematic representation of the Hib capsule polymer repeating unit. b, Genetic organization of region 2 of the capsule gene cluster of Hib. Schematic showing the following three predicted regions of the Bcs3 amino acid sequence: an N-terminal TagF-like protein, a central phosphatase protein and a C-terminal region containing a putative ribosyltransferase (RibT) protein. c, Schematic of an enzyme cascade for the synthesis of Hib polymer. Ribose-5-phosphate is converted by the d-ribose-5-phosphate isomerase RpiA into ribulose-5-phosphate, which is reduced (using NADPH) and activated (using CTP) by Bcs1 to yield CDP-ribitol, the donor substrate for the ribitol-5-phosphate transfer catalyzed by Bcs3. PRPP is the donor substrate for the ribose transfer catalyzed by Bcs3. Red and orange background colors refer to the moieties that are most likely transferred by the domains shown in b. d, HPLC-based AEC for the analysis of enzyme reactions as indicated by numbers in gray circles. The CMP peak in chromatogram 6 was cropped to harmonize the overall appearance of the figure. CDP-glycerol was used as a control for the commercially unavailable CDP-ribitol. e, Chemical structure of PRPP. f, Alcian blue-stained polyacrylamide gel corresponding to d. g, Dot blot analysis used a Hib-specific agglutination serum for detection. Phosphate-containing polymer from Neisseria meningitidis serogroup A⁵⁰ was used as negative control. h, The overall structure of the Bcs3 dimer in complex with CMP, with both protomers shown in surface representation (left) and one protomer shown in cartoon representation (right) to visualize the secondary structural organization and the CMP (green). Each protomer of the homodimer is composed of (1) the ribofuranosyltransferase CriT (red), (2) the phosphatase CrpP (yellow), (3) the ribitol-phosphate transferase CroT (orange) and (4) an SH3b domain (tan). See also Supplementary Fig. 5.

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