Abstract
Capsule of Escherichia coli O5:K4:H4 is formed of a chondroitin-repeat disaccharide unit of glucuronic acid (GlcA)-N-acetylgalactosamine (GalNAc). This polysaccharide, commonly referred to as K4CP, is a potentially important source of precursors for chemoenzymatic or bioengineering synthesis of chondroitin sulfate. KfoA, encoded by a gene from region 2 of the K4 capsular gene cluster, shows high homology to the UDP-glucose-4-epimerase (GalE) from E. coli. KfoA is reputed to be responsible for uridine 5′-diphosphate-N-acetylgalactosamine (UDP-GalNAc) supply for K4CP biosynthesis in vivo, but it has not been biochemically characterized. Here, we probed the substrate specificity of KfoA by a capillary electrophoresis (CE)-based method. KfoA could epimerize both acetylated and non-acetylated substrates, but its k cat/K m value for UDP-GlcNAc was approximately 1300-fold that for UDP-Glc. Recombinant KfoA showed a strong preference for acetylated substrates in vitro. The conclusion that KfoA is a higher efficiency UDP-GalNAc provider than GalE was supported by a coupled assay developed based on the donor-acceptor combination specificity of E. coli K4 chondroitin polymerase (KfoC). Furthermore, residue Ser-301, located near the UDP-GlcNAc binding pocket, plays an important role in the determination of the conversion ratio of UDP-GlcNAc to UDP-GalNAc by KfoA. Our results deepen the understanding of the mechanism of KfoA and will assist in the research into the metabolic engineering for chondroitin sulfate production.
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Acknowledgements
This work was supported in part by the National Natural Science Foundation of China (Project no. 31770845), as well as the Projects of the Science and Technology Department of Shandong Province (Project nos. 2015GSF121002 and 2015ZDJS04002). We gratefully thank Dr. Lan Jin for the discussion and suggestions.
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JS and FW designed and coordinated the work. HZ carried out the experiments. YL expressed the KfoA. HZ and DM prepared the KfoA mutants. HZ and TW prepared the donors. HZ and BS conducted the docking analysis. SZ conducted the MS analysis. JS and HZ wrote the manuscript. All authors read and approved the final manuscript.
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Zhu, HM., Sun, B., Li, YJ. et al. KfoA, the UDP-glucose-4-epimerase of Escherichia coli strain O5:K4:H4, shows preference for acetylated substrates. Appl Microbiol Biotechnol 102, 751–761 (2018). https://doi.org/10.1007/s00253-017-8639-0
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DOI: https://doi.org/10.1007/s00253-017-8639-0