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A gene encoding a dye-linked d-amino acid dehydrogenase (Dye-DADH) homologue was found in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. The predicted amino acid sequence suggested that the gene product is a membrane-bound type enzyme. The gene was overexpressed in Escherichia coli, but the recombinant protein was exclusively produced as an inclusion body. In order to avoid production of the inclusion body, an expression system using the thermoacidophilic archaeon Sulfolobus acidocaldarius instead of E. coli as the host cell was constructed. The gene was successfully expressed in Sulfolobus acidocaldarius, and its product was purified to homogeneity and characterized. The purified enzyme catalyzed the dehydrogenation of various d-amino acids, with d-phenylalanine being the most preferred substrate. The enzyme retained its full activity after incubation at 90 °C for 30 min and after incubation at pH 4.0-11.0 for 30 min at 50 °C. This is the first report on membrane-bound Dye-DADH from thermophilic archaea that was successfully expressed in an archaeal host., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A stable d-lactate electrochemical sensing system was developed using a dye-linked d-lactate dehydrogenase (Dye-DLDH) from an uncultivated thermophilic archaeon, Candidatus Caldiarchaeum subterraneum. To develop the system, the putative gene encoding the Dye-DLDH from Ca. Caldiarchaeum subterraneum was overexpressed in Escherichia coli, and the expressed product was purified. The recombinant enzyme was a highly thermostable Dye-DLDH that retained full activity after incubation for 10 min at 70°C. The electrode for detection of d-lactate was prepared by immobilizing the thermostable Dye-DLDH and multi-walled carbon nanotube (MWCNT) within Nafion membrane. The electrocatalytic response of the electrode was clearly observed upon exposure to d-lactate. The electrode response to d-lactate was linear within the concentration range of 0.03-2.5 mM, and it showed little reduction in responsiveness after 50 days. This is the first report describing a d-lactate sensing system using a thermostable Dye-DLDH., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Dye-linked D-amino acid dehydrogenases (Dye-DADHs) catalyze the dehydrogenation of free D-amino acids in the presence of an artificial electron acceptor. Although Dye-DADHs functioning in catabolism of L-alanine and as primary enzymes in electron transport chains are widely distributed in mesophilic Gram-negative bacteria, biochemical and biotechnological information on these enzymes remains scanty. This is in large part due to their instability after isolation. On the other hand, in the last decade, several novel types of Dye-DADH have been found in thermophilic bacteria and hyperthermophilic archaea, where they contribute not only to L-alanine catabolism but also to the catabolism of other amino acids, including D-arginine and L-hydroxyproline. In this minireview, we summarize recent developments in our understanding of the biochemical characteristics of Dye-DADHs and their specific application to electrochemical biosensors.

NAD(P)-dependent dehydrogenases differ according to their coenzyme preference: some prefer NAD, others NADP, and still others exhibit dual cofactor specificity. The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP. However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation. Structural analysis and site-directed mutagenesis showed that the large number of interactions between the cofactor and the enzyme are responsible for the lack of reactivity of the enzyme towards NADP. This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity.

Dye-linked L-proline dehydrogenase (ProDH) catalyzes the oxidation of L-proline to ∆(1)-pyrroline-5-carboxylate (P5C) in the presence of artificial electron acceptors. The enzyme is known to be widely distributed in bacteria and eukarya, together with nicotinamide adenine dinucleotide (phosphate)-dependent P5C dehydrogenase, and to function in the metabolism of L-proline to L-glutamate. In addition, over the course of the last decade, three other types of ProDH with molecular compositions completely different from previously known ones have been identified in hyperthermophilic archaea. The first is a heterotetrameric αβγδ-type ProDH, which exhibits both ProDH and reduced nicotinamide adenine dinucleotide dehydrogenase activity and includes two electron transfer proteins. The second is a heterooctameric α(4)β(4)-type ProDH, which uses flavin adenine dinucleotide, flavin mononucleotide, adenosine triphosphate, and Fe as cofactors and creates a new electron transfer pathway. The third is a recently identified homodimeric ProDH, which exhibits the greatest thermostability among these archaeal ProDHs. This minireview focuses on the functional and structural properties of these three types of archaeal ProDH and their distribution in archaea. In addition, we will describe the specific application of hyperthermostable ProDH for use in a biosensor and for DNA sensing.

The crystal structure of ST1625p, a protein encoded by a hypothetical open reading frame ST1625 in the genome of the hyperthermophilic archaeon Sulfolobus tokodaii, was determined at 2.2 A resolution. The only sequence similarity exhibited by the amino-acid sequence of ST1625p was a 33% identity with the sequence of SSO0983p from S. solfataricus. The 19 kDa monomeric protein was observed to consist of a right-handed superhelix assembled from a tandem repeat of ten alpha-helices. A structural homology search using the DALI and MATRAS algorithms indicates that this protein can be classified as a helical repeat protein.

Hyperthermophiles are a group of microorganisms that have their optimum growth temperature above 80 degrees C. More than 60 species of the hyperthermophiles have been isolated from marine and continental volcanic environments. Most hyperthermophiles belong to Archaea, the third domain of life, and are considered to be the most ancient of all extant life forms. Recent studies have revealed the presence of unusual sugar metabolic processes in hyperthermophilic archaea, for example, a modified Embden-Meyerhof pathway, that has so far not been observed in bacteria and eucarya. Several novel enzymes, such as ADP-dependent glucokinase, ADP-dependent phosphofructokinase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase, phosphoenolpyruvate synthase, pyruvate : ferredoxin oxidoreductase, and ADP-forming acetyl-CoA synthetase, have been found to be involved in a modified Embden-Meyerhof pathway of the hyperthermophilic archaeon Pyrococcus furiosus. In addition, a unique mode of ATP regeneration has been postulated to exist in the pathway of P. furiosus. The metabolic design observed in this microorganism might reflect the situation at an early stage of evolution., (Copyright 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.)

A gene encoding a 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homolog was identified in the hyperthermophilic Archaea Aeropyrum pernix. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. The enzyme is an extremely thermostable DERA; its activity was not lost after incubation at 100 degrees C for 10 min. The enzyme has a molecular mass of approximately 93 kDa and consists of four subunits with an identical molecular mass of 24 kDa. This is the first report of the presence of tetrameric DERA. The three-dimensional structure of the enzyme was determined by x-ray analysis. The subunit folds into an alpha/beta-barrel. The asymmetric unit consists of two homologous subunits, and a crystallographic 2-fold axis generates the functional tetramer. The main chain coordinate of the monomer of the A. pernix enzyme is quite similar to that of the E. coli enzyme. There was no significant difference in hydrophobic interactions and the number of ion pairs between the monomeric structures of the two enzymes. However, a significant difference in the quaternary structure was observed. The area of the subunit-subunit interface in the dimer of the A. pernix enzyme is much larger compared with the E. coli enzyme. In addition, the A. pernix enzyme is 10 amino acids longer than the E. coli enzyme in the N-terminal region and has an additional N-terminal helix. The N-terminal helix produces a unique dimer-dimer interface. This promotes the formation of a functional tetramer of the A. pernix enzyme and strengthens the hydrophobic intersubunit interactions. These structural features are considered to be responsible for the extremely high stability of the A. pernix enzyme. This is the first description of the structure of hyperthermophilic DERA and of aldolase from the Archaea domain.

A gene encoding galactose 1‐phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate‐free enzyme at 2.33 Å and the UDP‐bound H140F mutant enzyme at 1.78 Å. The main‐chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N‐terminal chain extends from one subunit into the other and forms part of the substrate‐binding pocket in the neighboring subunit. By contrast, the N‐terminal chain in P. aerophilum GalT extends to the substrate‐binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N‐terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate‐free enzyme with UDP‐bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction. [ABSTRACT FROM AUTHOR]

A gene encoding l-serine dehydrogenase (l-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciensl-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosal-SerDH, suggesting that P. calidifontisl-SerDH is a novel type of l-SerDH, like Ps. aeruginosal-SerDH. The overexpressed enzyme appears to be the most thermostable l-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards d-serine, in addition to l-serine. Two different crystal structures of P. calidifontisl-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP+/sulfate ion at 1.18 Å and the structure in complex with NADP+/l-tartrate (substrate analog) at 1.57 Å. The fold of the catalytic domain showed similarity with that of Ps. aeruginosal-SerDH. However, the active site structure significantly differed between the two enzymes. Based on the structure of the tartrate, l- and d-serine and 3-hydroxypropionate molecules were modeled into the active site and the substrate binding modes were estimated. A structural comparison suggests that the wide cavity at the substrate binding site is likely responsible for the high reactivity of the enzyme toward both l- and d-serine enantiomers. This is the first description of the structure of the novel type of l-SerDH with bound NADP+ and substrate analog, and it provides new insight into the substrate binding mechanism of l-SerDH. The results obtained here may be very informative for the creation of l- or d-serine-specific SerDH by protein engineering. [ABSTRACT FROM AUTHOR]

ABSTRACT A gene encoding an sn-glycerol-1-phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate ( Vmax) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2' of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild-type enzyme with NADPH, which suggests that the biased interactions around the C2'-phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)-dependent dehydrogenases. Proteins 2016; 84:1786-1796. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The cover image, by Haruhiko Sakuraba et al., is based on the Article Unique coenzyme binding mode of hyperthermophilic archaeal sn ‐glycerol‐1‐phosphate dehydrogenase from Pyrobaculum calidifontis, DOI: 10.1002/prot.25161. [ABSTRACT FROM AUTHOR]

A gene encoding NAD(P)H-dependent carbonyl reductase (CR) from the hyperthermophilic archaeon Aeropyrum pernix K1 was overexpressed in Escherichia coli . Its product was effectively purified and characterized. The expressed enzyme was the most thermostable CR found to date; the activity remained at approximately 75% of its activity after incubation for 10 min up to 90 °C. In addition, A. pernix CR exhibited high stability at a wider range of pH values and longer periods of storage compared with CRs previously identified from other sources. A. pernix CR catalyzed the reduction of various carbonyl compounds including ethyl 4-chloro-3-oxobutanoate and 9,10-phenanthrenequinone, similar to the CR from thyroidectomized (Tx) chicken fatty liver. However, A. pernix CR exhibited significantly higher K m values against several substrates than Tx chicken fatty liver CR. The three-dimensional structure of A. pernix CR was determined using the molecular replacement method at a resolution of 2.09 Å, in the presence of NADPH. The overall fold of A. pernix CR showed moderate similarity to that of Tx chicken fatty liver CR; however, A. pernix CR had no active-site lid unlike Tx chicken fatty liver CR. Consequently, the active-site cavity in the A. pernix CR was much more solvent-accessible than that in Tx chicken fatty liver CR. This structural feature may be responsible for the enzyme’s lower affinity for several substrates and NADPH. The factors contributing to the much higher thermostability of A. pernix CR were analyzed by comparing its structure with that of Tx chicken fatty liver CR. This comparison showed that extensive formation of the intrasubunit ion pair networks, and the presence of the strong intersubunit interaction, is likely responsible for A. pernix CR thermostability. Site-directed mutagenesis showed that Glu99 plays a major role in the intersubunit interaction. This is the first report regarding the characteristics and three-dimensional structure of hyperthermophilic archaeal CR. [ABSTRACT FROM AUTHOR]

Abstract: The crystal structure of a highly thermostable UDP-galactose 4-epimerase (GalE) from the hyperthermophilic archaeon Pyrobaculum calidifontis was determined at a resolution of 1.8Å. The asymmetric unit contained one subunit, and the functional dimer was generated by a crystallographic two-fold axis. Each monomer consisted of a Rossmann-fold domain with NAD bound and a carboxyl terminal domain. The overall structure of P. calidifontis GalE showed significant similarity to the structures of the GalEs from Escherichia coli, human and Trypanosoma brucei. However, the sizes of several surface loops were markedly smaller in P. calidifontis GalE than the corresponding loops in the other enzymes. Structural comparison revealed that the presence of an extensive hydrophobic interaction at the subunit interface is likely the main factor contributing to the hyperthermostability of the P. calidifontis enzyme. Within the NAD-binding site of P. calidifontis GalE, a loop (NAD-binding loop) tightly holds the adenine ribose moiety of NAD. Moreover, a deletion mutant lacking this loop bound NAD in a loose, reversible manner. Thus the presence of the NAD-binding loop in GalE is largely responsible for preventing the release of the cofactor from the holoenzyme. [Copyright &y& Elsevier]

The crystal structure of an extremely thermostable multicopper oxidase (McoP) from the hyperthermophilic archaeon Pyrobaculum aerophilum was determined at a resolution of 2.0 Å. The overall fold was comprised of three cupredoxin-like domains and the main-chain coordinates of the enzyme were similar to those of multicopper oxidases from Escherichia coli (CueO) and Bacillus subtilis (CotA). However, there were clear topological differences around domain 3 between McoP and the other two enzymes: a methionine-rich helix in CueO and a protruding helix in CotA were not present in McoP. Instead, a large loop (PL-1) covered the T1 copper centre of McoP and a short α-helix in domain 3 extended near the N-terminal end of PL-1. In addition, the sizes of several surface loops in McoP were markedly smaller than the corresponding loops in CueO and CotA. Structural comparison revealed that the presence of extensive hydrophobic interactions and a smaller cavity volume are likely to be the main factors contributing to the hyperthermostability of McoP. [ABSTRACT FROM AUTHOR]

Abstract: We identified a gene encoding a soluble quinoprotein glucose dehydrogenase homologue in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The enzyme was extremely thermostable, and the activity of the pyrroloquinoline quinone (PQQ)-bound holoenzyme was not lost after incubation at 100°C for 10min. The crystal structure of the enzyme was determined in both the apoform and as the PQQ-bound holoenzyme. The overall fold of the P. aerophilum enzyme showed significant similarity to that of soluble quinoprotein aldose sugar dehydrogenase (Asd) from E. coli. However, clear topological differences were observed in the two long loops around the PQQ-binding sites of the two enzymes. Structural comparison revealed that the hyperthermostability of the P. aerophilum enzyme is likely attributable to the presence of an extensive aromatic pair network located around a β-sheet involving N- and C-terminal β-strands. [ABSTRACT FROM AUTHOR]

Abstract: The crystal structure of the highly thermostable l-aspartate oxidase (LAO) from the hyperthermophilic archaeon Sulfolobus tokodaii was determined at a 2.09 Å resolution. The factors contributing to the thermostability of the enzyme were analyzed by comparing its structure to that of Escherichia coli LAO. Like E. coli LAO, the S. tokodaii enzyme consists of three domains: an FAD-binding domain, an α+β capping domain, and a C-terminal three-helix bundle. However, the situation of the linker between the FAD-binding domain and C-terminal three-helix bundle in S. tokodaii LAO is completely different from that in E. coli LAO, where the linker is situated near the FAD-binding domain and has virtually no interaction with the rest of the protein. In S. tokodaii LAO, this linker is situated near the C-terminal three-helix bundle and contains a β-strand that runs parallel to the C-terminal strand. This results in the formation of an additional β-sheet, which appears to reduce the flexibility of the C-terminal region. Furthermore, the displacement of the linker enables formation of a 5-residue ion-pair network between the FAD-binding and C-terminal domains, which strengthens the interdomain interactions. These features might be the main factors contributing to the high thermostability of S. tokodaii LAO. [Copyright &y& Elsevier]

Abstract: A gene (ST1218) encoding a d-3-phosphoglycerate dehydrogenase (PGDH; EC 1.1.1.95) homolog was found in the genome of Sulfolobus tokodaii strain 7 by screening a database of enzymes likely to contribute to l-serine biosynthesis in hyperthermophilic archaea. After expressing the gene in Escherichia coli, the PGDH activity of the recombinant enzyme was assessed. Homogeneous PGDH was obtained using conventional chromatography steps, though during the purification an unexpected decline in enzyme activity was observed if the enzyme was stored in plastic tubes, but not in glass ones. The purified enzyme was a homodimer with a subunit molecular mass of about 35kDa and was highly thermostable. It preferably acted as an NAD-dependent d-3-phosphoglycerate (3PGA) dehydrogenase. Although NADP had no activity as the electron acceptor, both NADPH and NADH acted as electron donors. Kinetic analyses indicated that the enzyme reaction proceeds via a Theorell-Chance Bi-Bi mechanism. Unlike E. coli PGDH, the S. tokodaii enzyme was not inhibited by l-serine. In addition, both the NAD-dependent 3PGA oxidation and the reverse reaction were enhanced by phosphate and sulfate ions, while NADPH-dependent 3-phosphohydroxypyruvate (PHP) reduction was inhibited. Thus S. tokodaii PGDH appears to be subject to a novel regulatory mechanism not seen elsewhere. A database analysis showed that ST1218 gene forms a cluster with ST1217 gene, and a functional analysis of the ST1217 product expressed in E. coli revealed that it possesses l-glutamate-PHP aminotransferase activity. Taken together, our findings represent the first example of a phosphorylated serine pathway in a hyperthermophilic archaeon. [Copyright &y& Elsevier]

Abstract: Agmatinase is one of the key enzymes in the biosynthesis of polyamines such as putrescine and sperimidine from arginine in microorganisms. The gene (PH0083) encoding the putative agmatinase of hyperthermophilic archaeon Pyrococcus horikoshii was identified based on the genome database. The gene was cloned and expressed, and the product was mainly obtained as inactive inclusion body in Escherichia coli. The inclusion body was dissolved in 6 M guanidine–HCl and successively refolded to active enzyme by the dilution of the denaturant. The enzyme exclusively catalyzed the hydrolysis of agmatine, but not arginine. This indicates that PH0083 codes agmatinase. The enzyme required divalent cations such as Co2+, Ca2+ and Mn2+ for the activity. The highest activity was observed under fairly alkaline conditions, like pH 11. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of 110–145 kDa. The enzyme was extremely thermostable: the full activity was retained on heating at 80 °C for 10 min, and a half of the activity was retained by incubation at 90 °C for 10 min. From a typical Michaelis–Menten type kinetics, an apparent K m value for agmatine was determined to be 0.53 mM. Phylogenic analysis revealed that the agmatinase from P. horikoshii does not belong to any clusters of enzymes found in bacteria and eukarya. This is the first description of the presence of archaeal agmatinase and its characteristics. [Copyright &y& Elsevier]

Oxidative stress response in an anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was analyzed by two-dimensional gel electrophoresis. When P. horikoshii was grown on medium supplemented with air, a marked increase in the level of a 25-kDa protein was observed in comparison with cells grown under anaerobic conditions. The N-terminal amino acid sequence of the protein was determined to be VVIGEKFPEVEVKTTHGVIKLPDYF, which coincides with that of the putative alkyl hydroperoxide reductase that has been predicted in the genome database of P. horikoshii. The gene (PH1217) encoding the protein was cloned and expressed in Escherichia coli. The produced enzyme was a hyperthermostable peroxiredoxin whose activity was not lost after incubation at 90°C for 20 min. The enzyme catalyzes the reduction of cumene hydroperoxide and hydrogen peroxide using dithiothreitol as an electron donor. Northern blot analysis revealed that the transcription of the gene increased by the addition of exogenous oxygen and by the addition of an oxidative stress-inducing reagent, and reached maximum within 30 min. These results suggest that the peroxiredoxin plays an important role in the peroxide-scavenging system in an anaerobic archaeon P. horikoshii. [ABSTRACT FROM PUBLISHER]

A gene encoding a nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT, EC 2.7.7.1) homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. The gene encoded a protein of 186 amino acids with a molecular weight of 21,391. The deduced amino acid sequence of the gene showed 59% identities to the NMNAT from Methanococcus jannaschii. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified to homogeneity. Characterization of the enzyme revealed that it is an extremely thermostable NMNAT; the activity was not lost after incubation at 80 °C for 30 min. The native molecular mass was estimated to be 77 kDa. The Km values for ATP and NMN were calculated to be 0.056 and 0.061 mM, respectively. The optimum temperature of the reaction was estimated to be around 90 °C. The adenylyl group donor specificity was examined by high-performance liquid chromatography (HPLC). At 70 °C, ATP was a prominent donor. However, above 80 °C, a relatively small, but significant, NMNAT activity was detected when ATP was replaced by ADP or AMP in the reaction mixture. To date, an NMNAT that utilizes ADP or AMP as an adenylyl group donor has not been found. The present study provides interesting information in which a di- or mono-phosphate nucleotide can be utilized by adenylyltransferase at high temperature. [Copyright &y& Elsevier]

A gene encoding the L-aspartate oxidase homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. We succeeded in expressing the encoding gene in Escherichia coli and purified the product to homogeneity. Characterization of the protein revealed that it is the most thermostable L-aspartate oxidase detected so far. In addition to the oxidase activity, the enzyme catalyzed L-aspartate dehydrogenation in the presence of an artificial electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, and ferricyanide. L-Aspartate oxidase is known to function as the first enzyme in the de novo NAD biosynthetic pathway in prokaryotes. By a similarity search in public databases, the genes that encode the homologue of all other enzymes involved in the pathway were identified in the P. horikoshii OT-3 genome. This suggests that P. horikoshii OT-3 may use the de novo NAD biosynthetic pathway under anaerobic conditions. [ABSTRACT FROM AUTHOR]

Hyperthermophiles, a group of microorganisms whose optimum growth temperatures are above 80°C, have been isolated mainly from marine and continental volcanic environments. They are viewed as potential sources of extraordinarily stable biomolecules with applications in novel industrial processes. Most hyperthermophiles belong to the domain Archaea, the third domain of life, and are considered to be the most ancient of all extant life forms. Recent studies have revealed unusual energy metabolic processes in hyperthermophilic archaea, e.g. a modified Embden-Meyerhof pathway, that have not been observed so far in organisms belonging to the Bacteria and Eucarya domains.Several novel enzymes — ADP-dependent glucokinase, ADP-dependent phosphofruktokinase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase, phosphoenolpyruvate synthase, pyruvate: ferredoxin oxidoreductase, and ADP-forming acetyl-CoA synthetase — have been found to be involved in the modified Embden-Meyerhof pathway of the hyperthermophilic archaeon Pyrococcus furiosus. In addition, a novel regulation site for energy metabolism and a unique mode of ATP regeneration have been postulated to exist in the pathway of P. furiosus. The metabolic design observed in this microorganism might reflect the situation at an early stage of evolution. This review focuses mainly on the unique energy metabolism and related enzymes of P. furiosus that have recently been described. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ornithine δ-aminotransferase (Orn-AT) activity was detected for the enzyme annotated as a γ-aminobutyrate aminotransferase encoded by PH1423 gene from Pyrococcus horikoshii OT-3. Crystal structures of this novel archaeal ω-aminotransferase were determined for the enzyme in complex with pyridoxal 5′-phosphate (PLP), in complex with PLP and l -ornithine (l -Orn), and in complex with N -(5′-phosphopyridoxyl)- l -glutamate (PLP- l -Glu). Although the sequence identity was relatively low (28%), the main-chain coordinates of P. horikoshii Orn-AT monomer showed notable similarity to those of human Orn-AT. However, the residues recognizing the α-amino group of l -Orn differ between the two enzymes. In human Orn-AT, Tyr55 and Tyr85 recognize the α-amino group, whereas the side chains of Thr92* and Asp93*, which arise from a loop in the neighboring subunit, form hydrogen bonds with the α-amino group of the substrate in P. horikoshii enzyme. Site-directed mutagenesis suggested that Asp93* plays critical roles in maintaining high affinity for the substrate. This study provides new insight into the substrate binding of a novel type of Orn-AT. Moreover, the structure of the enzyme with the reaction-intermediate analogue PLP- l -Glu bound provides the first structural evidence for the "Glu switch" mechanism in the dual substrate specificity of Orn-AT. • The crystal structures of a novel archaeal type of ornithine δ-aminotransferase were determined. • The substrate recognition residues of this enzyme are notably different from those of human ornithine δ-aminotransferase. • This study provides the first structural evidence for the "Glu switch" mechanism of ornithine δ-aminotransferase. [ABSTRACT FROM AUTHOR]

2-Deoxy- d -ribose-5-phosphate aldolase (DERA) catalyzes the aldol reaction between two aldehydes and is thought to be a potential biocatalyst for the production of a variety of stereo-specific materials. A gene encoding DERA from the extreme halophilic archaeon, Haloarcula japonica , was overexpressed in Escherichia coli . The gene product was successfully purified, using procedures based on the protein’s halophilicity, and characterized. The expressed enzyme was stable in a buffer containing 2 M NaCl and exhibited high thermostability, retaining more than 90% of its activity after heating at 70 °C for 10 min. The enzyme was also tolerant to high concentrations of organic solvents, such as acetonitrile and dimethylsulfoxide. Moreover, H. japonica DERA was highly resistant to a high concentration of acetaldehyde and retained about 35% of its initial activity after 5-h’ exposure to 300 mM acetaldehyde at 25 °C, the conditions under which E. coli DERA is completely inactivated. The enzyme exhibited much higher activity at 25 °C than the previously characterized hyperthermophilic DERAs (Sakuraba et al ., 2007). Our results suggest that the extremely halophilic DERA has high potential to serve as a biocatalyst in organic syntheses. This is the first description of the biochemical characterization of a halophilic DERA. [ABSTRACT FROM AUTHOR]

Abstract: A gene encoding an l-aspartate dehydrogenase (EC 1.4.1.21) homologue was identified in the anaerobic hyperthermophilic archaeon Archaeoglobus fulgidus. After expression in Escherichia coli, the gene product was purified to homogeneity, yielding a homodimeric protein with a molecular mass of about 48 kDa. Characterization revealed the enzyme to be a highly thermostable l-aspartate dehydrogenase, showing little loss of activity following incubation for 1 h at up to 80 °C. The optimum temperature for l-aspartate dehydrogenation was about 80 °C. The enzyme specifically utilized l-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor. The K m values for l-aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor. The K m values for NAD and NADP were 0.11 and 0.32 mM, respectively. For reductive amination, the K m values for oxaloacetate, NADH and ammonia were 1.2, 0.014 and 167 mM, respectively. The enzyme showed pro-R (A-type) stereospecificity for hydrogen transfer from the C4 position of the nicotinamide moiety of NADH. This is the first report of an archaeal l-aspartate dehydrogenase. Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species. [Copyright &y& Elsevier]