Genes encoding 2-deoxy-D-ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, were expressed individually in Escherichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E. coli enzyme, even at a low temperature (25°C), although both enzymes showed much less 2-deoxy-D-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25°C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 Å. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs.Using acetaldehyde and D-glyceraldehyde-3-phosphate as substrates, 2-deoxy-D-ribose-5-phosphate aldolase (DERA; EC 4.1.2.4) catalyzes a reversible aldol reaction that generates 2-deoxy-D-ribose-5-phosphate (DRP) (1,17). DERA is unique in that it catalyzes the aldol reaction between two aldehydes, which serve as both the aldol donor and the acceptor components. In addition, DERA is the only aldolase known to accept three aldehydes in a sequential and stereoselective manner during an aldol condensation reaction, which makes it a particularly interesting potential biocatalyst for synthetic organic chemistry. Gijsen and Wong (6) were the first to observe Escherichia coli DERA (DERA Eco ) catalyze a double aldol condensation of three acetaldehyde molecules (Fig. 1): the reaction started with a stereospecific addition of acetaldehyde to a substituted acetaldehyde to form 3-hydroxy-4-substituted butylaldehyde, which in turn reacted with a third acetaldehyde. After the second condensation, the product (compound 1) largely cyclized to form stable 2,4,6-trideoxy-D-erythro-hexapyranoside, which is a useful chiral synthon of hydroxymethylglutaryl-coenzyme A reductase inhibitors. This has prompted investigation of the feasibility of applying DERA Eco for the synthesis of cholesterol-lowering agents (5-7, 23). The practical application of th...

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...

Dye-linked l-proline dehydrogenase catalyzes the oxidation of l-proline in the presence of artificial electron acceptors such as 2, 6-dichloroindophenol and ferricyanide. The enzyme from the hyperthermophilic archaeon Thermococcus profundus was purified and characterized for the first time in archaea by Sakuraba et al. in 2001. In this study, cloning and sequencing analyses of the gene encoding the enzyme and functional analysis of the subunits were performed. The gene formed an operon that consisted of four genes, pdhA, pdhB, pdhF, and pdhX, which are tandemly arranged in the order of pdhA-F-X-B. SDS-PAGE analysis of the purified recombinant enzyme showed four different bands corresponding to alpha (54 kDa), beta (43 kDa), gamma (19 kDa), and delta (8 kDa) subunits encoded by pdhA, pdhB, pdhF, and pdhX, respectively, and the molecular ratio of these subunits was determined to be equal. This indicates that the enzyme consists of a heterotetrameric alphabetagammadelta structure. Functional analysis of each subunit revealed that the beta subunit catalyzed the dye-linked l-proline dehydrogenase reaction by itself and that, unexpectedly, the alpha subunit exhibited dye-linked NADH dehydrogenase activity. This is the first example showing the existence of a bifunctional dye-linked l-proline/NADH dehydrogenase complex. On the basis of genome analysis, similar gene clusters were observed in the genomes of Pyrococcus horikoshii, Pyrococcus abyssi, Pyrococcus furiosus, and Archaeoglobus fulgidus. These results indicate that the dye-linked l-proline dehydrogenase is a novel type of heterotetrameric amino acid dehydrogenase that might be widely distributed in the hyperthermophilic archaeal strain.

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Two novel types of dye-linked L-proline dehydrogenase complex (PDH1 and PDH2) were found in a hyperthermophilic archaeon, Pyrococcus horikoshii OT3. Here we report the first crystal structure of PDH1, which is a heterooctameric complex (␣␤) 4 containing three different cofactors: FAD, FMN, and ATP. The structure was determined by x-ray crystallography to a resolution of 2.86 Å. The structure of the ␤ subunit, which is an L-proline dehydrogenase catalytic component containing FAD as a cofactor, was similar to that of monomeric sarcosine oxidase. On the other hand, the ␣ subunit possessed a unique structure composed of a classical dinucleotide fold domain with ATP, a central domain, an N-terminal domain, and a Cys-clustered domain. Serving as a third cofactor, FMN was located at the interface between the ␣ and ␤ subunits in a novel configuration. The observed structure suggests that FAD and FMN are incorporated into an electron transfer system, with electrons passing from the former to the latter. The function of ATP is unknown, but it may play a regulatory role. Although the structure of the ␣ subunit differs from that of the ␤ subunit, except for the presence of an analogous dinucleotide domain with a different cofactor, the structural characteristics of PDH1 suggest that each represents a divergent enzyme that arose from a common ancestral flavoenzyme and that they eventually formed a complex to gain a new function. The structural characteristics described here reveal the PDH1 complex to be a unique diflavin dehydrogenase containing a novel electron transfer system. Dye-linked dehydrogenases catalyze the oxidation of various amino acids, organic acids, amines, and alcohols in the presence of an artificial electron acceptor such as ferricyanide or 2,6-dichloroindophenol (DCIP). 1 We have identified several novel dye-linked dehydrogenases among the hyperthermophilic archaea, including D-proline dehydrogenase (1) and Lproline dehydrogenase (PDH) (2, 3), which catalyzes the dehydrogenation from L-prroline to ⌬ 1 -pyrroline-5-carboxylate (P5C). The gene sequence and primary structure of the first PDH isolated from a hyperthermophilic archaeon, Thermococcus profundus DSM9503, have been determined (2, 3). The complete gene is formed by an operon comprised of four genes, pdhA, pdhB, pdhF and pdhX, in a tandem arrangement in the order pdhA-F-X-B. The purified enzyme has a native molecular mass of 120 kDa and forms an unusual ␣␤␥␦ heterotetrameric complex (␣ (54 kDa), ␤ (43 kDa), ␥ (19 kDa), and ␦ (11 kDa)) encoded by pdhA, pdhB, pdhF, and pdhX, respectively. In addition, functional analysis of each subunit has shown that the ␣ and ␤ subunits possess NADH and L-proline dehydrogenase activities, respectively (3). Because this multifunctional dye-linked dehydrogenase complex appears to representative of a new enzyme group (3), we have been seeking others by screening dye-linked dehydrogenases expressed in hyperthermophilic archaea. So far, we have identified two different types of dye-linked PDH in the hyperthermophilic ar...