Streptococcus pneumoniae is a major human pathogen associated with diseases worldwide. The capsular polysaccharides (CPSs) are considered a major virulence factor and are targets for a vaccine. d-Mannitol was found to be present in the CPS of several S. pneumoniae serotypes. Two genes, mnp1 and mnp2, which are located in the CPS gene cluster, were proposed to be responsible for the synthesis of NDP-d-mannitol (the nucleotide activated form of d-mannitol). However, the pathway has never been identified by experimental methods and we aimed to characterize it in the present study. To achieve this, the two genes, mnp1 and mnp2, were cloned and the gene products were overexpressed, purified, and analyzed in vitro for their respective enzymatic activities. Products of reactions catalyzed by Mnp1 and Mnp2 were detected by capillary electrophoresis and validated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. We show that Mnp1 is responsible for the transfer of CMP from CTP to d-fructose-6-phosphate (Fru-6-P) to form CDP-d-fructose, whereas Mnp2 catalyzed the conversion of CDP-d-fructose to CDP-d-mannitol. Therefore, Mnp1 (renamed as mnpA) was identified as Fru-6-P cytidylyltransferase-encoding gene, and mnp2 (renamed as mnpB) as a CDP-d-fructose reductase-encoding gene. The kinetics of Mnp1 for the substrate (Fru-6-P and CTP) and of Mnp2 for the substrate (CDP-d-fructose) and the cofactor NADH or NADPH fitted the Michaelis-Menten model. The effects of temperature, pH and cations on the two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-d-mannitol has been identified biochemically.

CDP-D-mannitol, cytidyltransferase, eductase

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
5,0,0	xDMan-ol	68.8	69.7	70.7	70.6	72.3	64.6
5,0	P
5	P
	xXnucC


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
5,0,0	xDMan-ol	4.14-4.21	3.89	3.88	3.81	3.78	3.68-3.87
5,0	P
5	P
	xXnucC


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
5,0,0	xDMan-ol	68.8/4.14-4.21	69.7/3.89	70.7/3.88	70.6/3.81	72.3/3.78	64.6/3.68-3.87
5,0	P
5	P
	xXnucC

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

Nc1ccn([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)O[C@H](CO)[C@@H](O)[C@H](O)[C@H](O)CO)[C@@H](O)[C@H]2O)c(=O)n1

567.334 g/mol (C₁₅H₂₇N₃O₁₆P₂)