Misincorporation of Galactose by Chondroitin Synthase of Escherichia coli K4: From Traces to Synthesis of Chondbiuronan, a Novel Chondroitin-Like Polysaccharide

Chondroitin synthase KfoC is a bifunctional enzyme which polymerizes the capsular chondroitin backbone of K4, composed of repeated β3N-acetylgalactosamine (GalNAc)-β4-glucuronic acid (GlcA) units. Sugar donors UDP-GalNAc and UDP-GlcA are the natural precursors of bacterial chondroitin synthesis. We...

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Published inBiomolecules (Basel, Switzerland) Vol. 10; no. 12; p. 1667
Main Authors Leroux, Mélanie, Michaud, Julie, Bayma, Eric, Armand, Sylvie, Drouillard, Sophie, Priem, Bernard
Format Journal Article
LanguageEnglish
Published Switzerland MDPI AG 12.12.2020
MDPI
Subjects
Bacteriology
Biotechnology
Chemical Sciences
chondroitin
Chromatography
Cloning
Cytoplasm
Deoxyribonucleic acid
DNA
E coli
Enzymes
Epimerase
Escherichia coli
Galactose
Genes
Genomics
Glycerol
K4 chondroitin synthase
Lactose
Life Sciences
Microbiology and Parasitology
Molecular weight
NDP-sugar misincorporation
NMR
Nuclear magnetic resonance
Polymers
Polysaccharides
Sweden
France
United States > US
Germany
chondroitin
NDP-sugar misincorporation
K4 chondroitin synthase
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Summary:Chondroitin synthase KfoC is a bifunctional enzyme which polymerizes the capsular chondroitin backbone of K4, composed of repeated β3N-acetylgalactosamine (GalNAc)-β4-glucuronic acid (GlcA) units. Sugar donors UDP-GalNAc and UDP-GlcA are the natural precursors of bacterial chondroitin synthesis. We have expressed KfoC in a recombinant strain of deprived of 4-epimerase activity, thus incapable of supplying UDP-GalNAc in the bacterial cytoplasm. The strain was also co-expressing mammal galactose β-glucuronyltransferase, providing glucuronyl-lactose from exogenously added lactose, serving as a primer of polymerization. We show by the mean of NMR analyses that in those conditions, KfoC incorporates galactose, forming a chondroitin-like polymer composed of the repeated β3-galactose (Gal)-β4-glucuronic acid units. We also show that when UDP-GlcNAc 4-epimerase KfoA, encoded by the K4-operon, was co-expressed and produced UDP-GalNAc, a small proportion of galactose was still incorporated into the growing chain of chondroitin.
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ISSN:2218-273X
2218-273X
DOI:10.3390/biom10121667