Molecular and biochemical analysis of MalK, the ATP-hydrolyzing subunit of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis.
We report the cloning, sequencing, and expression of malK encoding the ATP-hydrolyzing subunit of the maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis. According to the deduced amino acid sequence, MalK consists of 372 amino acids with a calculated molecular weight of 41,787. It shows 47% identity with the MalK protein of Escherichia coli and high sequence conservation in important regions. C-terminal His-tagged MalK was purified. The soluble protein appeared monomeric by molecular sieve chromatography and showed ATPase activity. Enzymatic activity was highest at 80 degrees C with a Km of 150 microM and a Vmax of 0.55 micromol of ATP hydrolyzed/min/mg of protein. ADP was not a substrate but a competitive inhibitor (Ki 230 microM). GTP and CTP were also hydrolyzed. ATPase activity was inhibited by N-ethylmaleimide but not by vanadate. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the ABC transporters in these two phylogenetic branches.
Greller
G
G
Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Horlacher
R
R
DiRuggiero
J
J
Boos
W
W
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019712:Thermococcus / Q000378:metabolism*; D014199:Trehalose / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; D011994:Recombinant Proteins; D008320:Maltose; D000255:Adenosine Triphosphate; D014199:Trehalose; D000251:Adenosine Triphosphatases