The sugar capsule capsular polysaccharide A (CPSA), which coats the surface of the mammalian symbiont Bacteroides fragilis, is a key mediator of mammalian immune system development. In addition, this sugar polymer has shown therapeutic potential in animal models of multiple sclerosis and other autoimmune disorders. The structure of the CPSA polymer includes a rare stereoconfiguration sugar acetamido-4-amino-6-deoxygalactopyranose (AADGal) that we propose is the first sugar linked to a bactoprenyl diphosphate scaffold in the production of CPSA. In this report, we have utilized a heterologous system to reconstitute bactoprenyl diphosphate-linked AADGal production. Construction of this system included a previously reported Campylobacter jejuni dehydratase, PglF, coupled to a B. fragilis-encoded aminotransferase (WcfR) and initiating hexose-1-phosphate transferase (WcfS). The function of the aminotransferase was confirmed by capillary electrophoresis and a novel high-performance liquid chromatography (HPLC) method. Production of the rare uridine diphosphate (UDP)-AADGal was confirmed through a series of one- and two-dimensional nuclear magnetic resonance experiments and high-resolution mass spectrometry. A spectroscopically unique analogue of bactoprenyl phosphate was utilized to characterize the transfer reaction catalyzed by WcfS and allowed HPLC-based isolation of the isoprenoid-linked sugar product. Importantly, the entire heterologous system was utilized in a single-pot reaction to biosynthesize the bactoprenyl-linked sugar. This work provides the first critical step in the in vitro reconstitution of CPSA biosynthesis.

Bacteroides fragilis, aminotransferase, caspsular polysaccharide A, biosynthesis pathway, WcfR, WcfS, acetamido-4-amino-6-deoxy-galactopyranose

13C NMR data:
missing...
1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
5,0,0,2	Ac	-	1.95
5,0,0,4	Ac
5,0,0	aDQuipN4N	5.38	3.99	3.82	2.99	4.19	1.26
5,0	P
5	P
	xXnucU

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O)[C@@H](NC(C)=O)[C@@H](OP(=O)(O)OP(=O)(O)OC[C@H]2O[C@@H](n3ccc(=O)[nH]c3=O)[C@H](O)[C@@H]2O)O[C@@H]1C

632.409 g/mol (C₁₉H₃₀N₄O₁₆P₂)