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KMID : 1094720060110040344
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Biotechnology and Bioprocess Engineering
2006 Volume.11 No. 4 p.344 ~ p.350
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Purification and characterization of glycerate kinase from the thermoacidophilic archaeon Thermoplasma acidophilum : An enzyme belonging to the second glycerate kinase family
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Noh Mi-Young
Jung Jin-Hwa
Lee Sun-Bok
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Abstract
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Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at 59°C and pH 2. Along with another thermoacidophilic archaeon,Sulfolobus solfataricus, it is known to metabolize glucose by the non-phosphorylated Entner-Doudoroff (nED) pathway. In the course of these studies, the specific activities of glyceraldehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher inT. acidophilum than inS. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity fromT. acidophilum cell extracts. TheN-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49 kDa and followed Michaelis-Menten kinetics withK m values of 0.56 and 0.32 mM forDL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at 70°C. Of the seven sugars and four phosphate donors tested, onlyDL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as Mn2+, Co2+, Ni2+, Zn2+, Ca2+, and Sr2+, were substituted for Mg2+, the enzyme activities were less than 10% of that obtained in the presence of Mg2+. The amino acid sequence ofT. acidophilum glycerate kinase showed no similarity withE. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.
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KEYWORD
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glycerate kinase, 2-phosphoglycerate, non-phosphorylated Entner-Doudoroff pathway, thermoacidophilic archaea, Thermoplasma acidophilum
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