title: Discovery and characterization of a novel ATP/polyphosphate xylulokinase from a hyperthermophilic bacterium Thermotoga maritima.
abstract: Xylulokinase (XK, E.C. 2.7.1.17) 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.
authors.affiliation: Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, VA 24061, USA.
authors.forename: Julia S
authors.initials: JS
authors.lastname: Martín del Campo
authors.forename: You
authors.initials: Y
authors.lastname: Chun
authors.forename: Jae-Eung
authors.initials: JE
authors.lastname: Kim
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Patiño
authors.forename: Y-H Percival
authors.initials: YH
authors.lastname: Zhang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002415:Cats; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006019:Glycolysis; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011122:Polyphosphates / Q000378:metabolism*; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014994:Xylose / Q000378:metabolism; D014996:Xylulose / Q000378:metabolism
chemical_list: D011122:Polyphosphates; D014996:Xylulose; D000255:Adenosine Triphosphate; D014994:Xylose; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase