Biosynthesis of dermatan sulphate. Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the D-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism.
The capsular polysaccharide from Escherichia coli K4 consists of a chondroitin ([GlcA(beta 1-->3)GalNAc(beta 1-->4)]n) backbone, to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid (GlcA) residues. Removal of the fructose units by mild acid hydrolysis provided a substrate for the GlcA C-5 epimerase, which is involved in the generation of L-iduronic acid (IdoA) units during dermatan sulphate biosynthesis. Incubation of this substrate with solubilized fibroblast microsomal enzyme in the presence of 3H2O resulted in the incorporation of tritium at C-5 of hexuronyl units. A Km of 67 x 10(-6) M hexuronic acid (equivalent to disaccharide units) was determined, which is similar to that (80 x 10(-6) M) obtained for dermatan (desulphated dermatan sulphate). Vmax was about 4 times higher with dermatan than with the K4 substrate. A defructosylated K4 polysaccharide isolated after incubation of bacteria with D-[5-3H]glucose released 3H2O on reaction with the epimerase, and thus could be used to assay the enzyme. Incubation of a K4 substrate with solubilized microsomal epimerase for 6 h in the presence of 3H2O resulted in the formation of about 5% IdoA and approximately equal amounts of 3H in GlcA and IdoA. A corresponding incubation of dermatan yielded approx. 22% GlcA, which contained virtually all the 3H label. These results are tentatively explained in terms of a two-base reaction mechanism, involving a monoprotic L-ido-specific base and a polyprotic D-gluco-specific base. Most of the IdoA residues generated by the enzyme occurred singly, although some formation of two or three consecutive IdoA-containing disaccharide units was observed.
Hannesson
H H
HH
Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
Hagner-McWhirter
A
A
Tiedemann
K
K
Lindahl
U
U
Malmström
A
A
D016667:Bacterial Capsules; D002238:Carbohydrate Epimerases / Q000378:metabolism; D002240:Carbohydrate Sequence; D003871:Dermatan Sulfate / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D005632:Fructose / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D013379:Substrate Specificity
D011135:Polysaccharides, Bacterial; C063323:capsular polysaccharide K4; D003871:Dermatan Sulfate; D005632:Fructose; D002238:Carbohydrate Epimerases; C019591:heparosan N-sulfate D-glucuronosyl 5-epimerase