Molecular and Biochemical Analysis of MalK, the ATP-hydrolyzing Subunit of the Trehalose/Maltose Transport System of the Hyperthermophilic Archaeon Thermococcus litoralis

Abstract

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°C with a K m of 150 M and a V max of 0.55 mol of ATP hydrolyzed/min/mg of protein. ADP was not a substrate but a competitive inhibitor (K i 230 M). 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.High affinity binding protein-dependent ABC transporters were originally discovered in Gram-negative bacteria. They consist of a binding protein as their major substrate recognition site, located in the periplasm, two hydrophobic membrane proteins forming the translocation pore, and, peripherally associated with them at the inner face of the membrane, two additional subunits. By ATP hydrolysis the latter provide the energy for the accumulation of substrate (1). In the case of the Escherichia coli maltose/maltodextrin transport system, malE is the gene for the periplasmic-binding protein (MBP or MalE), 1 the membrane components are encoded by genes malF and malG, and the two ATP-hydrolyzing subunits, by malK. These genes form a cluster on the E. coli chromosome where malE malF malG constitute an operon that is oriented divergently to malK (2). Binding protein-dependent ABC transporters have also been found in thermophilic bacteria (3, 4). Recently, we described an ABC transporter for maltose/trehalose in the hyperthermophilic archaeon Thermococcus litoralis (5). This transport system has several unusual properties. It shows an extremely high affinity (K m of about 20 nM) at 85°C, the optimum growth temperature of this organism, and it recognizes with equal affinity its very different substrates, maltose and trehalose, and is not inhibited by maltodextrins. The soluble high affinity maltose/trehalose-binding protein (TMBP) is anchored to the membrane by a lipid anchor. malE, the gene encoding TMBP, is the first gene in an operon with malF and malG as distal genes (6). Here we report the molecular characterization of malK and the biochemical properties of the encoded purified His-tagged protein. MATERIALS AND METHODS Cloning of T. litoralis malK-Chromosomal DNA was prepared fromT. litoralis by the method of Owen and Borman (7). Because the trehalose/maltose system of T. litoralis is nearly identical to that of Pyrococcus furiosus, we searched the P. fur...

The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis at 1.85 Å

“…8,42 is very similar to that of E. coli and other bacterial binding proteindependent (BPD) ABC transport systems. 1,43 In E. coli, the maltose transport system is in fact a maltodextrin transport system optimized for the utilization of maltose as well as of short maltodextrins.…”

Sugar transport in (hyper)thermophilic archaea

“…In contrast to the other proposed dimer organizations, the Rad50cd dimer model explains the results obtained from the mutational analysis in different ATP binding proteins [26]. It is also surprising that Tl-MalK crystalizes in a dimeric form, because the protein has been shown with several techniques to be a monomer in solution [11]. It is possible that for correct dimerization the contact with the membrane components of the complex is necessary.…”

Characterization of a Thermophilic P-type Ag+/Cu+-ATPase from the ExtremophileArchaeoglobus fulgidus

“…Considering the particular lipid composition of archaea membranes (34,35), it could be hypothesized that thermophilic membrane proteins should have particular functional and structural characteristics. However, although some A1A0-ATPases (the archaeal homologs of F 1 F 0 -ATPases), redox, and electron transport components from archaea have been studied (36,37), only sugar transporter ABC ATPases from Thermococcus litoralis (38,39) and Sulfolobus acidocaldarius (40) have been initially characterized. Thus, little information is available on the structure and function of thermophilic solute transport proteins.…”