期刊
BIOMOLECULES
卷 10, 期 12, 页码 -出版社
MDPI
DOI: 10.3390/biom10121667
关键词
chondroitin; K4 chondroitin synthase; NDP-sugar misincorporation
资金
- CIFRE fellowship from ANRT (Association Nationale de la Recherche et de la Technologie) [2016/0814]
- Labex ARCANE
- CBH-EUR-GS [ANR-17-EURE-0003]
- Glyco@Alps [ANR-15-IDEX-02]
- PolyNat Carnot Institut [ANR-16-CARN-0025-01]
Chondroitin synthase KfoC is a bifunctional enzyme which polymerizes the capsular chondroitin backbone of Escherichia coli K4, composed of repeated beta 3N-acetylgalactosamine (GalNAc)-beta 4-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 Escherichia coli deprived of 4-epimerase activity, thus incapable of supplying UDP-GalNAc in the bacterial cytoplasm. The strain was also co-expressing mammal galactose beta-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 beta 3-galactose (Gal)-beta 4-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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