Gene expression and characterization of 2-keto-3-deoxygluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of the aerobic and acidophilic hyperthermophile Sulfolobus tokodaii.
2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the ATP-dependent phosphorylation of 2-keto-3-deoxygluconate, a key intermediate in the modified (semi-phosphorylative) Entner-Doudoroff (ED) glucose metabolic pathway. We identified the gene (ORF ID: ST2478) encoding KDGK in the hyperthermophilic archaeon Sulfolobus tokodaii based on the structure of a gene cluster in a genomic database and functionally expressed it in Escherichia coli. The expressed protein was purified from crude extract by heat treatment and two conventional column chromatography steps, and the partial amino acid sequence in the N-terminal region of the purified enzyme (MAKLIT) was identical to that obtained from the gene sequence. The purified enzyme was extremely thermostable and retained full activity after heating at 80 degrees C for 1 h. The enzyme utilized ATP or GTP, but not ADP or AMP, as a phosphoryl donor and 2-keto-3-deoxy-D-gluconate or 2-keto-D-gluconate as a phosphoryl acceptor. Divalent cations including Mg(2+), Co(2+), Ni(2+), Zn(2+) or Mn(2+) were required for activity, and the apparent Km values for KDG and ATP at 50 degrees C were 0.027 mM and 0.057 mM, respectively. The presence of KDGK means that the hyperthermophilic archaeon S. tokodaii metabolizes glucose via both modified (semi-phosphorylative) and non-phosphorylative ED pathways.
Ohshima
Toshihisa
T
Microbial Genetics Division, Institute of Genetic Resources, Kyushu University, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-8581, Japan. ohshima@agr.kyushu-u.ac.jp
Kawakami
Ryushi
R
Kanai
Yuichiro
Y
Goda
Shuichiro
S
Sakuraba
Haruhiko
H
D019843:Archaeal Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D002413:Cations, Divalent / Q000737:chemistry; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011685:Purine Nucleotides / Q000737:chemistry; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D002413:Cations, Divalent; D008670:Metals; D011685:Purine Nucleotides; D017853:Phosphotransferases (Alcohol Group Acceptor); C021471:2-keto-3-deoxygluconokinase; D005947:Glucose