Noticeable points of our study are the first structure of ADP-dependent kinase, the structural similarity to members of the ATP-dependent ribokinase family, its rare nucleotide specificity caused by a shift in nucleotide binding position by one phosphate unit, and identification of the residues that discriminate ADP- and ATP-dependence. The strict conservation of the binding site for the terminal and adjacent phosphate moieties suggests a common ancestral origin of both the ATP- and ADP-dependent kinases.

The ADP-dependent (AMP-forming) glucokinases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis catalyze the phosphorylation of glucose using ADP as the essential phosphoryl group donor. Both enzymes were purified to homogeneity and characterized with regard to each other. The enzymes had similar enzymological properties as to substrate specificity, coenzyme specificity, optimum pH, and thermostability. However, a difference was observed in the subunit composition; while the T. litoralis enzyme is a monomer with a molecular mass of 52 kDa, the P. furiosus enzyme has a molecular mass of about 100 kDa and consists of two subunits with identical molecular masses of 47 kDa. The genes encoding these enzymes were cloned and sequenced. The gene for the P. furiosus enzyme contains an open reading frame for 455 amino acids with a molecular weight of 51,265, and that for the T. litoralis enzyme contains an open reading frame for 467 amino acids with a molecular weight of 53,621. About 59% similarity in amino acid sequence was observed between these two enzymes, whereas they did not show similarity with any ATP-dependent kinases that have been reported so far. In addition, two phosphate binding domains, and adenosine and glucose binding motifs commonly conserved in the eukaryotic hexokinase family were not observed.

A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The gene encoded a protein of 462 amino acids with a molecular weight of 53,361. The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase. A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric. The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate. The K m values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mM, respectively. The K m values for ADP were 0.032 and 0.63 mM when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively. The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase. A gene duplication event might lead to the two enzymatic activities.In general, ATP is regarded as the universal energy carrier and the most common phosphoryl group donor for kinases. However, several gluco-and phosphofructokinases have been reported to have different phosphoryl group donor specificity. The glucokinase from Mycobacterium tuberculosis can utilize both ATP and polyphosphate as the phosphoryl group donor (1). PP i -dependent phosphofructokinases have been reported to be present in several eucarya and bacteria and in the hyperthermophilic archaeon Thermoproteus tenax (2-4). Recently novel sugar kinases, ADP-dependent (AMP-forming) glucokinase (ADP-GK) 1 and phosphofructokinase (ADP-PFK), were discovered in the hyperthermophilic archaeon Pyrococcus furiosus (5). Those enzymes require ADP as the phosphoryl group donor instead of ATP and are involved in a modified EmbdenMeyerhof pathway in this organism. The hyperthermophilic archaea are relatively deeply branched archaea and are considered to be phylogenetically ancient organisms. Therefore, structural analysis of the kinases from these organisms may provide abundant information for phylogenetic analysis of the sugar kinases. We cloned and sequenced the gene encoding the ADP-GKs from P. furiosus and Thermococcus litoralis (The nucleotide sequences have been submitted to the GenBank TM data bases as the genes for ADP-dependent hexokinase and are available under accession numbers E14588 and E14589.) (6). About 59% identity in amino acid sequence was observed between these two enzymes, although they did not show similarity with any ATP-dependent kinases that have been reported so far. In addition, the amino acid sequence of the P. furiosus ADP-GK showed high identity (26%) with that reported for the P. furiosus ADP-PFK (7). This suggests that those kinases belong to a...

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