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Objective: The construction of a novel bioanode based on l-proline oxidation using a cascade reaction pathway comprised of thermostable dehydrogenases. Results: A novel multi-enzymatic cascade pathway, containing four kinds of dehydrogenases from thermophiles (dye-linked l-proline dehydrogenase, nicotinamide adenine dinucleotide (NAD)-dependent Δ1-pyrroline-5-carboxylate dehydrogenase, NAD-dependent l-glutamate dehydrogenase and dye-linked NADH dehydrogenase), was designed for the generation of six-electrons from one molecule of l-proline. The current density of the four-dehydrogenase-immobilized electrode, with a voltage of + 450 mV (relative to that of Ag/AgCl), was 226.8 μA/cm2 in the presence of 10 mM l-proline and 0.5 mM ferrocene carboxylate at 50 °C. This value was 4.2-fold higher than that of a similar electrode containing a single dehydrogenase. In addition, about 54% of the initial current in the multi-enzyme cascade bioanode was maintained even after 15 days. Conclusions: Efficient deep oxidation of l-proline by multiple-enzyme cascade reactions was achieved in our designed electrode. The multi-enzyme cascade bioanode, which was built using thermophilic dehydrogenases, showed high durability at room temperature. The long-term stability of the bioanode indicates that it shows great potential for applications as a long-lived enzymatic fuel cell. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A gene from the thermophilic Gram-negative bacterium Rhodothermus marinus JCM9785, encoding a dye-linked d-amino acid dehydrogenase homologue, was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was a highly thermostable dye-linked d-amino acid dehydrogenase that retained more than 80 % of its activity after incubation for 10 min at up to 70 °C. When enzyme-catalyzed dehydrogenation of several d-amino acids was carried out using 2,6-dichloroindophenol as the electron acceptor, d-phenylalanine was the most preferable substrate among the d-amino acids tested. Immediately upstream of the dye-linked d-amino acid dehydrogenase gene (dadh) was a gene encoding a 4-hydroxyproline 2-epimerase homologue (hypE). That gene was successfully expressed in E. coli, and the gene product exhibited strong 4-hydroxyproline 2-epimerase activity. Reverse transcription PCR and quantitative real-time PCR showed that the six genes containing the dadh and hypE genes were arranged in an operon and were required for catabolism of trans-4-hydroxy- l-proline in R. marinus. This is the first description of a dye-linked d-amino acid dehydrogenase (Dye-DADH) with broad substrate specificity involved in trans-4-hydroxy- l-proline catabolism. [ABSTRACT FROM AUTHOR]

Crystal structures of the thermostable meso-diaminopimelate dehydrogenase (DAPDH) from Ureibacillus thermosphaericus were determined for the enzyme in the apo form and in complex with NADP+ and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. The main-chain coordinates of the enzyme showed notable similarity to those of Symbiobacterium thermophilum DAPDH. However, the subunit arrangement of U. thermosphaericus DAPDH (a dimer) was totally different from that of the S. thermophilum enzyme (a hexamer). Structural comparison with the dimeric enzyme from the mesophile Corynebacterium glutamicum revealed that the presence of large numbers of intrasubunit and intersubunit hydrophobic interactions, as well as the extensive formation of intersubunit ion-pair networks, were likely to be the main factors contributing to the higher thermostability of U. thermosphaericus DAPDH. This differs from S. thermophilum DAPDH, within which the unique hexameric assembly is likely to be responsible for its high thermostability. Analysis of the active site of U. thermosphaericus DAPDH revealed the key factors responsible for the marked difference in substrate specificity between DAPDH and the D-amino acid dehydrogenase recently created from DAPDH by introducing five point mutations [Akita et al. (2012). Biotechnol. Lett. 34, 1693-1699; 1701-1702]. [ABSTRACT FROM AUTHOR]

d-isoleucine ( d-Ile) can be assayed using chiral chromatography but the availability of that method is limited by the necessity for special expertise and expensive equipment. We therefore developed a simple and specific colorimetric assay system for d-Ile determination using an artificially created NADP-dependent d-amino acid dehydrogenase (DAADH). The system consists of two reaction steps: the first is the quantitative conversion of d-Ile to (3 R)-2-oxo-3-methyl valerate by DAADH in which NADP is converted to NADPH, while the second is chemical conversion of NADPH to reduced water-soluble Tetrazolium-3 via a redox mediator. d-Ile was determined from 1 to 50 µM, and the assay was unaffected by the presence of any of three other isomers (100 µM), alcohol and organic acids. [ABSTRACT FROM AUTHOR]

d-Branched-chain amino acids ( d-BCAAs) such as d-leucine, d-isoleucine, and d-valine are known to be peptide antibiotic intermediates and to exhibit a variety of bioactivities. Consequently, much effort is going into achieving simple stereospecific synthesis of d-BCAAs, especially analogs labeled with stable isotopes. Up to now, however, no effective method has been reported. Here, we report the establishment of an efficient system for enantioselective synthesis of d-BCAAs and production of d-BCAAs labeled with stable isotopes. This system is based on two thermostable enzymes: d-amino acid dehydrogenase, catalyzing NADPH-dependent enantioselective amination of 2-oxo acids to produce the corresponding d-amino acids, and glucose dehydrogenase, catalyzing NADPH regeneration from NADP and d-glucose. After incubation with the enzymes for 2 h at 65°C and pH 10.5, 2-oxo-4-methylvaleric acid was converted to d-leucine with an excellent yield (>99 %) and optical purity (>99 %). Using this system, we produced five different d-BCAAs labeled with stable isotopes: d-[1-C,N]leucine, d-[1-C]leucine, d-[N]leucine, d-[N]isoleucine, and d-[N]valine. The structure of each labeled d-amino acid was confirmed using time-of-flight mass spectrometry and nuclear magnetic resonance analysis. These analyses confirmed that the developed system was highly useful for production of d-BCAAs labeled with stable isotopes, making this the first reported enzymatic production of d-BCAAs labeled with stable isotopes. Our findings facilitate tracer studies investigating d-BCAAs and their derivatives. [ABSTRACT FROM AUTHOR]

Two types of hetero-oligomeric dye-linked l-proline dehydrogenases (αβ and αβγδ types) are expressed in the hyperthermophilic archaea belonging to Thermococcales. In both enzymes, the β subunit (PDHβ) is responsible for catalyzing l-proline dehydrogenation. The genes encoding the two enzyme types form respective clusters that are completely conserved among Pyrococcus and Thermococcus strains. To compare the enzymatic properties of PDHβs from αβ- and αβγδ-type enzyme complexes, eight PDHβs (four of each type) from Pyrococcus furiosus DSM3638, Pyrococcus horikoshii OT-3, Thermococcus kodakaraensis KOD1 JCM12380 and Thermococcus profundus DSM9503 were expressed in Escherichia coli cells and purified to homogeneity using one-step Ni-chelating chromatography. The αβ-type PDHβs showed greater thermostability than most of the αβγδ-type PDHβs: the former retained more than 80 % of their activity after heating at 70 °C for 20 min, while the latter showed different thermostabilities under the same conditions. In addition, the αβ-type PDHβs utilized ferricyanide as the most preferable electron acceptor, whereas αβγδ-type PDHβs preferred 2, 6-dichloroindophenol, with one exception. These results indicate that the differences in the enzymatic properties of the PDHβs likely reflect whether they were from an αβγδ- or αβ-type complex, though the wider divergence observed within αβγδ-type PDHβs based on the phylogenetic analysis may also be responsible for their inconsistent enzymatic properties. By contrast, differences in the kinetic parameters among the PDHβs did not reflect the complex type. Interestingly, the k value for free αβ-type PDHβ from P. horikoshii was much larger than the value for the same subunit within the αβ-complex. This indicates that the isolated PDHβ could be a useful element for an electrochemical system for detection of l-proline. [ABSTRACT FROM AUTHOR]

A thermostable, NADP-dependent d-amino acid dehydrogenase (DAADH) was created from the meso-diaminopimelate dehydrogenase of Ureibacillus thermosphaericus strain A1 by introducing five point mutations into amino acid residues located in the active site. The recombinant protein, expressed in Escherichia coli, was purified to homogeneity using a two-step separation procedure and then characterized. In the presence of NADP, the protein catalyzed the oxidative deamination of several d-amino acids, including d-cyclohexylalanine, d-isoleucine and d-2-aminooctanoate, but not meso-diaminopimelate, confirming the creation of a NADP-dependent DAADH. For the reverse reaction, the corresponding 2-oxo acids were aminated in the presence of NADPH and ammonia. In addition, the d-amino acid dehydrogenase showed no loss of activity at 65 °C, indicating the mutant enzyme was more thermostable than its parental meso-diaminopimelate dehydrogenase. [ABSTRACT FROM AUTHOR]

The article discusses the identification of the homologue of the enzyme L-proline dehydrogenase (LPDH) from the hyperthermophilic archaeon Pyrobaculum calidifontis in the aerobic hyperthermophilic archaeon Aeropyrum pernix. The purified enzyme is characterized as a homodimeric structure of molecular mass of 88 kilo dalton with thermostability property. The structural analysis of enzymes for study of the evolution of LPDHs and amino acid metabolism in hyperthermophilic archaea is also analyzed.

Dye-linked l-proline dehydrogenase (ProDH) catalyzes the oxidation of l-proline to ∆-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 αβ-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. [ABSTRACT FROM AUTHOR]

The activity of a dye-linked l-proline dehydrogenase (dye- l-proDH) was found in the crude extract of an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis JCM 11548, and was purified 163-fold through four sequential chromatography steps. The enzyme has a molecular mass of about 108 kDa and is a homodimer with a subunit molecular mass of about 46 kDa. The enzyme retained more than 90% of its activity after incubation at 100 °C for 120 min (pH 7.5) or after incubation at pHs 4.5-9.0 for 30 min at 50 °C. The enzyme catalyzed l-proline dehydrogenation to Δ-pyroline-5-carboxylate using 2,6-dichloroindophenol (DCIP) as the electron acceptor and the Michaelis constants for l-proline and DCIP were 1.67 and 0.026 mM, respectively. The prosthetic group on the enzyme was identified as flavin adenine dinucleotide by high-performance liquid chromatography. The subunit N-terminal amino acid sequence was MYDYVVVGAG. Using that sequence and previously reported genome information, the gene encoding the enzyme (Pcal_1655) was identified. The gene was then cloned and expressed in Escherichia coli and found to encode a polypeptide of 415 amino acids with a calculated molecular weight of 46,259. The dye- l-proDH gene cluster in P. calidifontis inherently differs from those in the other hyperthermophiles reported so far. [ABSTRACT FROM AUTHOR]

The crystal structure of the highly thermostablel-aspartate dehydrogenase (l-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate. The dimeric structure of A. fulgidusl-aspDH was refined at a resolution of 1.9 Å with a crystallographic R-factor of 21.7% ( Rfree = 22.6%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. Structural comparison of the A. fulgidusl-aspDH/NAD/citrate ternary complex and the Thermotoga maritimal-aspDH/NAD binary complex showed that A. fulgidusl-aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding. Like T. maritimal-aspDH, the A. fulgidus enzyme is highly thermostable . But whereas a large number of inter- and intrasubunit ion pairs are responsible for the stability of A. fulgidusl-aspDH, a large number of inter- and intrasubunit aromatic pairs stabilize the T. maritima enzyme. Thus stabilization of these twol-aspDHs appears to be achieved in different ways. This is the first detailed description of substrate and coenzyme binding tol-aspDH and of the molecular basis of the high thermostability of a hyperthermophilicl-aspDH. [ABSTRACT FROM AUTHOR]

Abstract: l-Proline sensor has been fabricated through multilayer assembly of recombinant dye-linked l-proline dehydrogenase (l-proDH) originally from hyperthermophilic archaeon (Therococcus profundus) and polymerized mediator, poly(allylamine) ferrocene (PAA-Fc), on a gold electrode by electrostatic layer-by-layer alternate adsorption method. The characteristic redox peaks of ferrocene groups were obvious exhibited in cyclic voltammograms, and both anodic and cathodic peaks increased with PAA-Fc/l-proDH bilayer number which confirmed the formation of a multilayer structure. Further experiments showed that an electrode fabricated in this way catalyzed the oxidation of l-proline. It suggested that polymerized mediator could transfer electrons between electrode surface and immobilized l-proDH. Ampeormetric experiments showed that the current response to l-proline increased with PAA-Fc/l-proDH bilayer number. The stability of the sensor was measured, and it kept a 40% relative response after 30 days storage in buffer under 4°C. [Copyright &y& Elsevier]

Two distinguishable activity bands for dye-linked l-proline dehydrogenase (PDH1 and PDH2) were detected when crude extract of the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was run on a polyacrylamide gel. After purification, PDH1 was found to be composed of two different subunits with molecular masses of 56 and 43 kDa, whereas PDH2 was composed of four different subunits with molecular masses of 52, 46, 20 and 8 kDa. The native molecular masses of PDH1 and PDH2 were 440 and 101 kDa, respectively, indicating that PDH1 has an α4β4 structure, while PDH2 has an αβγδ structure. PDH2 was found to be similar to the dye-linked l-proline dehydrogenase complex from Thermococcus profundus, but PDH1 is a different type of enzyme. After production of the enzyme in Escherichia coli, high-performance liquid chromatography showed the PDH1 complex to contain the flavins FMN and FAD as well as ATP. Gene expression and biochemical analyses of each subunit revealed that the β subunit bound FAD and exhibited proline dehydrogenase activity, while the α subunit bound ATP, but unlike the corresponding subunit in the T. profundus enzyme, it exhibited neither proline dehydrogenase nor NADH dehydrogenase activity. FMN was not bound to either subunit, suggesting it is situated at the interface between the α and β subunits. A comparison of the amino-acid sequences showed that the ADP-binding motif in the α subunit of PDH1 clearly differs from that in the α subunit of PDH2. It thus appears that a second novel dye-linked l-proline dehydrogenase complex is produced in P. horikoshii. [ABSTRACT FROM AUTHOR]

A gene encoding the L-threonine dehydrogenase homologue has been identified in a hyperthermophlic archaeon Pyrococcus horikoshii OT3 via genome sequencing. The gene was cloned and expressed in Escherichia coli. The purified enzyme from the recombinant E. coli was extremely thermostable; the activity was not lost after incubation at 100°C for 20 min. The enzyme (molecular mass: 192 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme is specific for NAD and utilizes L-threonine, L-serine and DL- threo-3-phenylserine as the substrate. The enzyme required divalent cations such as Zn2+, Mn2+ and Co2+ for the activity, and contained one zinc ion/subunit. The Km values for L-threonine and NAD at 50°C were 0.20 mM and 0.024 mM, respectively. Kinetic analyses indicated that the L-threonine oxidation reaction proceeds via a random mechanism with regard to the binding of L-threonine and NAD. The enzyme showed pro- R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NADH. This is the first description of the characteristics of an L-threonine dehydrogenase from the archaea domain. [ABSTRACT FROM AUTHOR]

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 α (54 kDa), β (43 kDa), γ (19 kDa), and δ (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 αβγδ structure. Functional analysis of each subunit revealed that the β subunit catalyzed the dye-linked l-proline dehydrogenase reaction by itself and that, unexpectedly, the α 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. [ABSTRACT FROM AUTHOR]

L-Lysine dehydrogenase, which catalyzes the oxidative deamination of L-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Δ¹-piperideine-6-carboxylate, indicating that the enzyme is L-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70°C, respectively. No activity was lost at temperatures up to 65°C in the presence of 5 mM L-lysine. The enzyme was relatively selective for L-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for L-lysine, NAD, and NADP at 50°C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da. [ABSTRACT FROM AUTHOR]

Lecture dehydrogenase has been purified to homogeneity from a moderate thermophilic actinomycete, Thermoactinomyces intermedius IFO 14230. The enzyme can be stored without loss of its activity at a low temperature (e.g. 4°C) for over two years. The enzyme was more thermostable at higher concentrations of salts such as NaCl and KCl. It retained about 90% of activity on incubation at 70%C for at least 40 min in the presence of 3 M NaCl. The Michaelis constants for NAD, L-leucine, NADH, 2-oxoisocaproate and ammonia were determined to be 0.36, 2.0, 0.042, 0.63 and 118 mM, respectively, from initial-velocity analyses. The enzyme showed gene was cloned into Escherichia coli and its complete DNA sequence was determined. The leucine dehydrogenase gene (leudh) consists of a 1098-bp open reading frame and encodes 366 amino acid residues corresponding to a subunit (Mr 40586) of the octameric enzyme. The amino acid sequence of the enzyme showed 80.7% similarity with that of the Bacillus stearothermophilus enzyme. The enzyme was overproduced in E. coli JM 109 having a recombinant plasmid, pULDH2, which was constructed from pUC18 and the leudh gene. The enzyme was purified from the cell extract to homogeneity in one day, with 78% recovery. [ABSTRACT FROM AUTHOR]

Alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile. Bacillus sphaericus DSM 461, b ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme has a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75°C for at least 60 min and with incubation in pH 5.5-9.5 at 75°C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20°C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate. 2-oxobutryrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM. [ABSTRACT FROM AUTHOR]

The article focuses on a study that characterizes the uracil-disphosphate -glucose dehydrogenase (UDP-GDH) identified from the Hyperthermophilic Archaon, Pyrobaculum islandicum. It notes that the physiological functions of UDP-GDH have demonstrated its importance in polysaccharide biosynthesis, detoxification and embryonic development. Furthermore, the higher stability of P. islandicum UDP-GDH has been significant in preparing highly pure enzymes.

A novel dye-linked l-proline dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 8000 as the precipitant. The crystals belonged to the tetragonal space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 61.1, c = 276.3 Å, and diffracted to 2.87 Å resolution using a Cu Kα rotating-anode generator with an R-AXIS VII detector. The asymmetric unit contained one protein molecule, giving a crystal volume per enzyme mass ( VM) of 2.75 Å3 Da−1 and a solvent content of 55.3%. [ABSTRACT FROM AUTHOR]

NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum was crystallized in the apo- and holoenzyme forms. Crystals were obtained using 2-propanol and polyethylene glycol MME 550 as precipitants for the apoenzyme and holoenzyme, respectively. The apoenzyme crystals belong to the trigonal space group P3121 or its enantiomorph P3221. The asymmetric unit contains three subunits; the values of the Matthews coefficient (VM) and the solvent content are 2.9 ų Da-1 and 57%, respectively. A native data set was collected to a highest resolution limit of 4.0 Å on an in-house X-ray source using a rotating-anode generator (overall Rsym of 12.3% and completeness of 97%). the holoenzyme crystals belong to the orthorhombic space group P2-12-12-1; the asymmetric unit contains one hexamer, giving a VM of 2.79 &Aring³ Da-1 and a solvent content of 55%. Native and derivative data sets were collected. The crystals diffract to a maximum resolution of 2.8 Å on the KEK-NW12 beamline at the Photon Factory and gave a data set with an overall Rsym of 7.9% and a completeness of 91%. Attempts are being made to solve the structure by the SIRAS method. [ABSTRACT FROM AUTHOR]

Glutamate dehydrogenase from an aerobic hyperthermophilic archaeon, Aeropyrum pernix K1, was crystallized by the hangingdrop vapour-diffusion method using polyethylene glycol (PEG) 400 as the precipitant. The crystals belong to the hexagonal space group P[sub 63], with unit-cell parameters a = b = 98.9, c = 394.8 Å, α = β = 90, γ = 120°. The asymmetric unit contained one hexamer of the enzyme, giving a crystal volume per enzyme mass (V[sub M]) of 1.98 ų Da[sup -1] and a solvent content of 37.3%. The X-ray diffraction data were collected to a resolution of 3.0 Å at the BL6B beamline in the Photon Factory with an overall R[sub sym] of 13.8% and a completeness of 87.1%. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The extremely thermostable NAD-dependent glutamate dehydrogenase (NAD-GluDH) from Pyrobaculum islandicum, a member of the Crenarchaeota, was crystallized, and its 3D structure has been determined by X-ray diffraction methods. The homohexameric structure of Pb.islandicum glutamate dehydrogenase (Pis-GluDH) was solved and refined at a resolution of 2.9Å with a crystallographic R-factor of 19.9% (Rfree 26.0%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. The secondary structural elements and catalytically important residues of the enzyme were highly conserved among the NAD(P)-dependent GluDHs from other sources. A structural comparison of Pis-GluDH with other NAD(P)-dependent GluDHs suggests that a significant difference in the α8-loop-α9 region of this enzyme is associated with its coenzyme specificity. From the analysis of the 3D structure, hydrophobic interactions between intersubunits were found to be important features for the enzyme oligomerization. It has been reported that Pis-GluDH is highly thermostable, like the GluDH of the hyperthermophilic archaeum Pyrococcus furiosus, and the increase in the intersubunit ion pair networks is responsible for the extreme thermostability of the Pc.furiosus enzyme. However, the number of intersubunit ion pairs in the Pis-GluDH molecules is much smaller than those of the Pc.furiosus GluDH. The number of hydrophobic interactions at the intersubunit interfaces were increased and responsible for the extremely high thermostability. This indicates that the major molecular strategy for high thermostability of the GluDHs may be different for each hyperthermophile. [Copyright &y& Elsevier]

Two putative alanine dehydrogenase (AlaDH) genes (GK2752 and GK3448) were found in the genome of a thermophilic spore-forming bacterium, Geobacillus kaustophilus. The amino acid sequences deduced from the two genes showed mutually high homology (71%), and the phylogenetic tree based on the amino acid sequences of the two putative AlaDHs and the homologous proteins showed that the two putative AlaDH genes (GK2752 and GK3448) belong to different groups. Both of the recombinant gene products exhibited high NAD+-dependent AlaDH activity and were purified to homogeneity and characterized in detail. Both enzymes showed high stability against low and high pHs and high temperatures (70 °C). Kinetic analyses showed that the activities of both enzymes proceeded according to the same sequentially ordered Bi-Ter mechanism. X-ray crystallographic analysis showed the two AlaDHs to have similar homohexameric structures. Notably, GK3448-AlaDH was detected in vegetative cells of G. kaustophilus but not spores, while GK2752-AlaDH was present only in the spores. This is the first report showing the presence of two AlaDHs separately expressed in vegetative cells and spores. • Two putative alanine dehydrogenase genes are present in G. kaustophilus genome. • Both the gene products exhibit high alanine dehydrogenase (AlaDH) activities. • The two purified enzymes show similar catalytic properties and hexamer structures. • GK3448-AlaDH is detected in vegetative cells, while GK2752-AlaDH only in the spores. [ABSTRACT FROM AUTHOR]

Abstract: Leucine dehydrogenase (LeuDH, l-leucine: NAD+ oxidoreductase, deaminating, EC 1.4.1.9) was screened in six psychrophilic bacteria, and the highest levels of enzyme activity were found in Sporosarcina psychrophila DSM 3. As the first LeuDH from a psychrophilic bacterium, the enzyme was purified to homogeneity and characterized. The protein had an octameric structure with identical 43-kDa subunits, giving a total molecular mass of about 340kDa. The enzyme exhibited the highest activity at 50°C and exhibited one-tenth of that activity even at temperatures as low as 0°C. The enzyme lost no activity with incubation at temperatures lower than 40°C for 40min, but there was marked loss of activity with incubations at temperatures higher than 50°C. The optimum pHs were 11 for deamination of l-leucine and 9 for amination of 4-methyl-2-oxopentanoate. The K m values for l-leucine and NAD+ at 20°C were 0.65 and 0.015mM, respectively. The catalytic properties of S. psychrophila LeuDH were similar to those of LeuDHs from Lysinibacillus sphaericus and Geobacillus stearothermophilus, except for its lower optimal reaction temperature and thermostability at low temperatures. Crystal structural analysis of S. psychrophila LeuDH showed the total structure to be similar to that of the L. sphaericus enzyme, except minor alterations reduced the hydrophobic interactions and hydrogen bonds within and between subunits. [Copyright &y& Elsevier]

Two types of dye-linked L-proline dehydrogenase (PDH1, α4β4-type hetero-octamer, and PDH2, αβγδ-type heterotetramer) have been identified so far in hyperthermophilic archaea. Here, we report the crystal structure of a third type of L-proline dehydrogenase, found in the aerobic hyperthermophilic archaeon Aeropyrum pernix, whose structure (homodimer) is much simpler than those of previously studied L-proline dehydrogenases. The structure was determined at a resolution of 1.92 Å. The asymmetric unit contained one subunit, and a crystallographic 2-fold axis generated the functional dimer. The overall fold of the subunit showed similarity to that of the PDH1 β-subunit, which is responsible for catalyzing L-proline dehydrogenation. However, the situation at the subunit-subunit interface of the A. pernix enzyme was totally different from that in PDH1. The presence of additional surface elements in the A. pernix enzyme contributes to a unique dimer association. Moreover, the C-terminal Leu428, which is provided by a tail extending from the FAD-binding domain, shielded the active site, and an L-proline molecule was entrapped within the active site cavity. The Km value of a Leu428 deletion mutant for L-proline was about 800 times larger than the Km value of the wild-type enzyme, although the kcat values did not differ much between the two enzymes. This suggests the C-terminal Leu428 is not directly involved in catalysis, but it is essential for maintaining a high affinity for the substrate. This is the first description of an LPDH structure with L-proline bound, and it provides new insight into the substrate binding of LPDH. [ABSTRACT FROM AUTHOR]

A gene from the thermophilic archaeon Thermoplasma volcanium encoding an l-threonine dehydrogenase (l-ThrDH) with a predicted amino acid sequence that was remarkably similar to the sequence of UDP-galactose 4-epimerase (GalE) was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was moderately thermostable, retaining more than 90% of its activity after incubation for 10 min at up to 70 °C. The catalytic residue was assessed using site-directed mutagenesis, and Tyr137 was found to be essential for catalysis. To clarify the structural basis of the catalytic mechanism, four different crystal structures were determined using the molecular replacement method: l-ThrDH-NAD+, l-ThrDH in complex with NAD+ and pyruvate, Y137F mutant in complex with NAD+ and l-threonine, and Y137F in complex with NAD+ and l-3-hydroxynorvaline. Each monomer consisted of a Rossmann-fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed notable similarity to that of the GalE-like l-ThrDH from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of 1-threonine via direct proton transfer driven by Tyr137. The factors contributing to the thermostability of T. volcanium l-ThrDH were analyzed by comparing its structure to that of F. frigidimaris l-ThrDH. This comparison showed that the presence of extensive inter- and intrasubunit ion pair networks are likely responsible for the thermostability of T. volcanium l-ThrDH. This is the first description of the molecular basis for the substrate recognition and thermostability of a GalE-like 1-ThrDH. [ABSTRACT FROM AUTHOR]

Abstract: Methods with which to simply and rapidly assay l-aspartate (l-Asp) and d-aspartate (d-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of l- and d-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an l-aspartate dehydrogenase (l-AspDH) system to colorimetrically assay l-Asp and a system of three hyperthermophilic enzymes—aspartate racemase (AspR), l-AspDH, and l-aspartate oxidase (l-AO)—to assay d-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD+)-dependent l-AspDH was measured based on increases in the absorbance at 438nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, d-Asp was measured after first removing l-Asp in the sample solution with l-AO. The remaining d-Asp was then changed to l-Asp using racemase, and the newly formed l-Asp was assayed calorimetrically using NAD+-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100μM l- and d-Asp in the assay systems. In addition, methods were applicable to the l- and d-Asp determinations in some living cells and foods. [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]

A gene encoding an L-lysine dehydrogenase was identified in the hyperthermophilic archaeon Pyrococcus horikoshii. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme is the most thermostable L-lysine dehydrogenase yet described, with a half-life of 180 mm at 100 °C. The product of the enzyme's catalytic activity is Δ1-piperideine-6-carboxylate, which makes this enzyme an L-lysine 6-dehydrogenase (EC 1.4.1.18) that catalyzes the reductive deamination of the ϵ-amino group and a type of NAD-dependent amine dehydrogenase. The three-dimensional structure of the enzyme was determined using the mercury-based multiple-wavelength anomalous dispersion method at a resolution of 2.44 Å in the presence of NAD and sulfate ion. The asymmetric unit consisted of two subunits, and a crystallographic 2-fold axis generated the functional dimer. Each monomer consisted of a Rossmann fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed similarity to that of saccharopine reductase. Notably, the structures of subunits A and B differed significantly. In subunit A, the active site contained a sulfate ion that was not seen in subunit B. Consequently, subunit A adopted a closed conformation, whereas subunit B adopted an open one. In each subunit, one NAD molecule was bound to the active site in an anti-conformation, indicating that the enzyme makes use of pro-R-specific hydride transfer between the two hydrides at C-4 of NADH (type A specificity). This is the first description of the three-dimensional structure of L-lysine 6-dehydrogenase as an NAD-dependent amine dehydrogenase. [ABSTRACT FROM AUTHOR]

Dye-linked d-lactate dehydrogenase activity was found in the crude extract of a continental thermoacidophilic crenarchaeota, Sulfolobus tokodaii strain 7, and was purified 375-fold through four sequential chromatography steps. With a molecular mass of about 93 kDa, this enzyme was a homodimer comprised of identical subunits with molecular masses of about 48 kDa. The enzyme retained its full activity after incubation at 80°C for 10 min and after incubation at pHs ranging from 6.5 to 10.0 for 30 min at 50°C. The preferred substrate for this enzyme was d-lactate, with 2,6-dichloroindophenol serving as the electron acceptor. Using high-performance liquid chromatography (HPLC), the enzyme''s prosthetic group was determined to be flavin adenine dinucleotide (FAD). Its N-terminal amino acid sequence was MLEGIEYSQGEEREDFVGFKIKPKI. Using that sequence and previously reported genome information, the gene encoding the enzyme (ST0649) was identified. It was subsequently cloned and expressed in Escherichia coli and found to encode a polypeptide of 440 amino acids with a calculated molecular weight of 49,715. The amino acid sequence of this dye-linked d-lactate dehydrogenase showed higher homology (39% identity) with that of a glycolate oxidase subunit homologue from Archaeoglobus fulgidus, but less similarity (32% identity) to d-lactate dehydrogenase from A. fulgidus. Taken together, our findings indicate that the dye-linked d-lactate dehydrogenase from S. tokodaii is a novel type of FAD containing d-lactate dehydrogenase. [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: 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]

The specific activity of recombinant Pyrobaculum islandicum glutamate dehydrogenase (pis- GDH) expressed in Escherichia coli is much lower than that of the native enzyme. However, when the recombinant enzyme is heated at 90 °C or exposed to 5 M urea, the activity increases to a level comparable to that of the native enzyme. Small-angle X-ray scattering measurements revealed that the radius of gyration (Rg,z) of the hexameric recombinant enzyme was reduced to 47 Å from 55 Å by either heat or urea, and that the final structure of the active enzyme is the same irrespective of the mechanism of activation. Activation was accompanied by a shift in the peaks of the Kratky plot, though the molecular mass of the enzyme was unchanged. The activation-induced decline in Rg,z followed first-order kinetics, indicating that activation of the enzyme involved a transition between two states, which was confirmed by singular-value decomposition analysis. When the low-resolution structure of the recombinant enzyme was restored using ab initio modeling, we found it to possess no point symmetry, whereas the heat-activated enzyme possessed 32-point symmetry. In addition, a marked increase in the fluorescence emission was observed with addition of ANS to the inactive recombinant enzyme but not the active forms, indicating that upon activation hydrophobic residues on the surface of the recombinant protein moved to the interior. Taken together, these data strongly suggest that subunit rearrangement, i.e., a change in the quaternary structure of the hexameric recombinant pis-GDH, is essential for activation of the enzyme. [ABSTRACT FROM AUTHOR]

Two novel types of dye-linked L-praline 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 (αβ)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 β 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 a 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 α and β 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 a subunit differs from that of the β 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. [ABSTRACT FROM AUTHOR]