Identification of the Glucosamine Kinase in the Chitinolytic Pathway of Thermococcus Kodakarensis

J Biosci Bioeng. 2018 Mar;125(3):320-326. doi: 10.1016/j.jbiosc.2017.10.005. Epub 2017 Nov 14.

Abstract

Although the chitinolytic pathway of the hyperthermophilic archaeon Thermococcus kodakarensis is well-studied, the genome does not contain genes homologous to previously identified glucosamine kinase genes. As some ADP-dependent glucokinases in the order Thermococcales exhibit phosphorylation activities for both glucose and glucosamine in vitro, the homolog in T. kodakarensis, encoded by TK1110, was selected as a candidate for the missing glucosamine kinase gene. The purified, recombinant TK1110 enzyme exhibited phosphorylation activities for not only glucose but also glucosamine and N-acetylglucosamine. Kinetic analysis indicated that activity towards glucosamine was as significant as that towards glucose. In order to determine the physiological role of TK1110 in the chitinolytic pathway of T. kodakarensis, a gene disruption strain of TK1110 was constructed. When grown in chitin-containing medium, the TK1110 disruption resulted in almost complete impairment in chitin degradation, and a complete loss of chitin-dependent H2 production. As H2 production is tightly linked to cell growth in T. kodakarensis, the present results strongly suggest that TK1110 functions as the glucosamine kinase responsible for the chitin degradation in T. kodakarensis.

Keywords: ADP-dependent glucokinase; Chitinolytic pathway; Glucosamine kinase; Hyperthermophiles; Thermococcus kodakarensis.

MeSH terms

  • Chitin / metabolism*
  • Cloning, Molecular
  • Glucokinase / genetics
  • Glucokinase / metabolism
  • Glucosamine / metabolism*
  • Hydrolysis
  • Kinetics
  • Metabolic Networks and Pathways / genetics*
  • Phosphotransferases / genetics*
  • Phosphotransferases / isolation & purification
  • Thermococcus / enzymology*
  • Thermococcus / genetics
  • Thermococcus / metabolism

Substances

  • Chitin
  • Phosphotransferases
  • ADP-dependent glucokinase
  • Glucokinase
  • Glucosamine