Discovery and characterization of a novel ATP/polyphosphate xylulokinase from a hyperthermophilic bacterium Thermotoga maritima
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Xylulokinase (XK, E.C. 22.214.171.124) is one of the key enzymes in xylose metabolism and it is essential for the activation of pentoses for the sustainable production of biocommodities from biomass sugars. The open reading frame (TM0116) from the hyperthermophilic bacterium Thermotoga maritima MSB8 encoding a putative xylulokinase were cloned and expressed in Escherichia coli BL21 Star (DE3) in the Luria–Bertani and auto-inducing high-cell-density media. The basic biochemical properties of this thermophilic XK were characterized. This XK has the optimal temperature of 85 °C. Under a suboptimal condition of 60 °C, the k cat was 83 s−1, and the K m values for xylulose and ATP were 1.24 and 0.71 mM, respectively. We hypothesized that this XK could work on polyphosphate possibly because this ancestral thermophilic microorganism utilizes polyphosphate to regulate the Embden–Meyerhof pathway and its substrate-binding residues are somewhat similar to those of other ATP/polyphosphate-dependent kinases. This XK was found to work on low-cost polyphosphate, exhibiting 41 % of its specific activity on ATP. This first ATP/polyphosphate XK could have a great potential for xylose utilization in thermophilic ethanol-producing microorganisms and cell-free biosystems for low-cost biomanufacturing without the use of ATP.
KeywordsBiomanufacturing Cell-free biosystem In vitro synthetic biology Polyphosphate Thermoenzyme Thermotoga maritima Xylulokinase
This work was supported by the Biological Systems Engineering Department of Virginia Tech, and partially supported Shell by GameChanger Program, the CALS Biodesign and Bioprocessing Research Center, and NSF SBIR grant to PZ. JSMC thanks the Mexican Council of Science and Technology (Conacyt) for her PhD study.
- 2.Anderson RL, Wood WA (1961) Purification and properties of l-xylulokinase. J Biol Chem 237:1029–1033Google Scholar
- 15.Iwanoto S, Motomura K, Shinoda Y, Urata M, Kato J, Takiguchi N, Ohtake H, Hirota R, Kuroda A (2007) Use of an Escherichia coli recombinant producing thermostable polyphosphate kinase as an ATP regenerator to produce fructose 1,6-diphosphate. Appl Environ Microbiol 73(17):5676–5678CrossRefGoogle Scholar
- 26.Nelson DL, Cox MM (2008) Lehninger principles of biochemistry, 5th edn. WH Freeman, New YorkGoogle Scholar
- 27.Nelson KE, Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Nelson WC, Ketchum KA, McDonald L, Utterback TR, Malek JA, Linher KD, Garrett MM, Stewart AM, Cotton MD, Pratt MS, Phillips CA, Richardson D, Heidelberg J, Sutton GG, Fleischmann RD, Eisen JA, White O, Salzberg SL, Smith HO, Venter JC, Fraser CM (1999) Evidence for lateral gene transfer between Archaea and Bacteria from genome sequence of Thermotoga maritima. Nature 399(6734):323–329PubMedCrossRefGoogle Scholar