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Virus capsid proteins have various applications in diverse fields such as biotechnology, electronics, and medicine. In this study, the major capsid protein of bacilliform clavavitus APBV1, which infects the hyperthermophilic archaeon Aeropyrum pernix, was successfully expressed in Escherichia coli. The gene product was expressed as a histidine-tagged protein in E. coli and purified to homogeneity using single-step nickel affinity chromatography. The purified recombinant protein self-assembled to form bacilliform virus-like particles at room temperature. The particles exhibited tolerance against high concentrations of organic solvents and protein denaturants. In addition, we succeeded in fabricating functional nanoparticles with amine functional groups on the surface of ORF6-81 nanoparticles. These robust protein nanoparticles can potentially be used as a scaffold in nanotechnological applications. [ABSTRACT FROM AUTHOR]

When overexpressed as an immature enzyme in the mesophilic bacterium Escherichia coli, recombinant homoserine dehydrogenase from the hyperthermophilic archaeon Sulfurisphaera tokodaii (StHSD) was markedly activated by heat treatment. Both the apo- and holo-forms of the immature enzyme were successively crystallized, and the two structures were determined. Comparison among the structures of the immature enzyme and previously reported structures of mature enzymes revealed that a conformational change in a flexible part (residues 160–190) of the enzyme, which encloses substrates within the substrate-binding pocket, is smaller in the immature enzyme. The immature enzyme, but not the mature enzyme, formed a complex that included NADP+, despite its absence during crystallization. This indicates that the opening to the substrate-binding pocket in the immature enzyme is not sufficient for substrate-binding, efficient catalytic turnover or release of NADP+. Thus, specific conformational changes within the catalytic region appear to be responsible for heat-induced activation. Heat-induced maturation of homoserine dehydrogenase from a hyperthermophilic archaeon requires conformational changes within the catalytic region. [ABSTRACT FROM AUTHOR]

Enzymes from hyperthermophilic archaea are potential candidates for industrial use because of their superior pH, thermal, and long-term stability, and are expected to improve the long-term stability of biofuel cells (BFCs). However, the reported multicopper oxidase (MCO) from hyperthermophilic archaea has lower redox potential than MCOs from other organisms, which leads to a decrease in the cell voltage of BFCs. In this study, we attempted to positively shift the redox potential of the MCO from hyperthermophilic archaeon Pyrobaculum aerophilum (McoP). Mutations (M470L and M470F) were introduced into the axial ligand of the T1 copper atom of McoP, and the enzymatic chemistry and redox potentials were compared with that of the parent (M470). The redox potentials of M470L and M470F shifted positively by about 0.07 V compared with that of M470. In addition, the catalytic activity of the mutants towards 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) increased 1.2–1.3-fold. The thermal stability of the mutants and the electrocatalytic performance for O2 reduction of M470F was slightly reduced compared with that of M470. This research provides useful enzymes for application as biocathode catalysts for high-voltage BFCs. [ABSTRACT FROM AUTHOR]

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]

Indigo is an insoluble blue dye historically used for dyeing textiles. A traditional approach for indigo dyeing involves microbial reduction of polygonum indigo to solubilize it under alkaline conditions; however, the mechanism by which microorganisms reduce indigo remains poorly understood. Here, we aimed to identify an enzyme that catalyzes indigo reduction; for this purpose, from alkaline liquor that performed microbial reduction of polygonum indigo, we isolated indigo carmine-reducing microorganisms. All isolates were facultative anaerobic and alkali-tolerant Bacillus spp. An isolate termed AO1 was found to be an alkaliphile that preferentially grows at pH 9.0-11.0 and at 30-35 °C. We focused on flavin-dependent azoreductase as a possible enzyme for indigo carmine reduction and identified its gene (azoA) in Bacillus sp. AO1 using homology-based strategies. azoA was monocistronic but clustered with ABC transporter genes. Primary sequence identities were < 50% between the azoA product (AzoA) and previously characterized flavin-dependent azoreductases. AzoA was heterologously produced as a flavoprotein tolerant to alkaline and organic solvents. The enzyme efficiently reduced indigo carmine in an NADH-dependent manner and showed strict specificity for electron acceptors. Notably, AzoA oxidized NADH in the presence, but not the absence, of indigo. The reaction rate was enhanced by adding organic solvents to solubilize indigo. Absorption spectrum analysis showed that indigo absorption decreased during the reaction. These observations suggest that AzoA can reduce indigo in vitro and potentially in Bacillus sp. AO1. This is the first study that identified an indigo reductase, providing a new insight into a traditional approach for indigo dyeing. [ABSTRACT FROM AUTHOR]

A gene-encoding a dye-linked D-lactate dehydrogenase (Dye-DLDH) homolog was identified in the genome of the hyperthermophilic archaeon Thermoproteus tenax. The gene was expressed in Escherichia coli and the product was purified to homogeneity. The recombinant protein exhibited highly thermostable Dye-DLDH activity. To date, four types of Dye-DLDH have been identified in hyperthermophilic archaea (in Aeropyrum pernix, Sulfolobus tokodaii, Archaeoglobus fulgidus, and Candidatus Caldiarchaeum subterraneum). The amino acid sequence of T. tenax Dye-DLDH showed the highest similarity (45%) to A. pernix Dye-DLDH, but neither contained a known FAD-binding motif. Nonetheless, both homologs required FAD for enzymatic activity, suggesting that FAD binds loosely to the enzyme and is easily released unlike in other Dye-DLDHs. Our findings indicate that Dye-DLDHs from T. tenax and A. pernix are a novel type of Dye-DLDH characterized by loose binding of FAD. [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]

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]

To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of d-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using C-labeled d-Trp showed that d-Trp in the medium was used as a protein component in the cells, suggesting the presence of d-amino acid metabolic enzymes. Pyridoxal 5′-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with d-amino acids, especially d- allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of d-amino acids for growth. [ABSTRACT FROM AUTHOR]

Objectives: The life of biobatteries remains an issue due to loss of enzyme activity over time. In this study, we sought to develop a biobattery with a long life using a hyperthermophilic enzyme. Results: We hypothesized that use of such hyperthermophilic enzymes would allow for the biofuel cells to have a long battery life. Using pyrroloquinoline quinone-glucose dehydrogenase and the multicopper oxidase from Pyrobaculum aerophilum, we constructed an anode and cathode. The maximum output was 11 μW at 0.2 V, and the stability of the both electrode was maintained at 70 % after 14 days. Conclusion: The biofuel cells that use hyperthermophilic enzymes may prolong their life. [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]

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 putative glutamate dehydrogenase (GDH) genes ( pcal_1031 and pcal_1606) were found in a sulfur-dependent hyperthermophilic archaeon, Pyrobaculum calidifontis. The two genes were then expressed in Escherichia coli, and both of the recombinant gene products showed GDH activity. The two enzymes were then purified to homogeneity and characterized in detail. Although both purified GDHs had a hexameric structure and neither exhibited allosteric regulation, they showed different coenzyme specificities: one was specific for NAD, the other for NADP and different heat activation mechanisms. In addition, there was little difference in the kinetic constants, optimal temperature, thermal stability, optimal pH and pH stability between the two enzymes. The overall sequence identity between the two proteins was very high (81 %), but was not high in the region recognizing the 2′ position of the adenine ribose moiety, which is responsible for coenzyme specificity. This is the first report on the identification of two GDHs with different coenzyme specificities from a single hyperthermophilic archaeon and the definition of their basic in vitro properties. [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 sporulation inhibitory gene spi in the pock-forming conjugative plasmid pSA1.1 of Streptomyces azureus was introduced into cells via a high or low copy number vector to examine the effect of gene dosage on the growth of Streptomyces lividans TK24 as a host. In transformants carrying a high spi copy number, nutrient mycelial growth was inhibited, as was morphological differentiation from substrate mycelium to aerial mycelium on solid media. The degree of inhibition depended on the spi gene dosage, but the presence of pSA1.1 imp genes, which encode negative repressor proteins for spi, relieved the inhibition. Confocal images of Spi tagged with enhanced green fluorescent protein in cells on solid media revealed that spi expression was initiated at the time of elongation of substrate mycelium, that its expression increased dramatically at septation in aerial hyphae, and that the expression was maximal during prespore formation. Expression of spi covered the whole of the hyphae, and the level of expression at the tip of the hyphae during prespore formation was about sixfold greater than during substrate mycelial growth and threefold greater than during aerial mycelial growth. Thus, localized expression of spi at particular times may inhibit sporulation until triggering imp expression to repress its inhibitory effects. [ABSTRACT FROM AUTHOR]

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]

Peptide tags containing tyrosines (Y-tag) were introduced at the C-terminus of a hyperthermophilic enzyme, alkaline phosphatase from Pyrococcus furiosus (PfuAP). Immobilization of the recombinant PfuAPs onto water-in-oil-in-water (W/O/W) type microcapsules was performed by an in situ polymerization method. All the recombinant PfuAPs prepared in this study were quantitatively immobilized onto microcapsules. The PfuAP-immobilized microcapsules showed no significant loss of enzymatic activity until the 5th round of assays. This result implies that the recombinant PfuAPs were covalently immobilized onto microcapsules. Immobilized PfuAP tagged with a Y-tag having the sequence GGYYY exhibited approximately a twofold higher catalytic activity compared with the wild-type PfuAP. [ABSTRACT FROM AUTHOR]

We conducted a comparative analysis of the effects of beryllium fluoride (BeFx) on protein folding mediated by the α- and β-subunit homooligomers (α16mer or β16mer) from the hyperthermophilic archaeum Thermococcus strain KS-1. BeFx inhibited the ATPase activities of both α16mer and β16mer with equal efficiency. This indicated that BeFx replaces the γ-phosphate of chaperonin-bound ATP, thereby forming a stable chaperonin–ADP–BeFx complex. In the presence of ATP and BeFx, both of the two chaperonin subunits mediated green fluorescent protein (GFP) folding. Gel filtration chromatography revealed that the refolded GFP was retained by both chaperonins. Protease digestion and electron microscopic analyses showed that both chaperonin–ADP–BeFx complexes of the two subunits adopted a symmetric closed conformation with the built-in lids of both rings closed and that protein folding took place in their central cavities. These data indicated that basic protein folding mechanisms of α16mer and β16mer are likely similar although there were some apparent differences. While β16mer-mediated GFP refolding in the presence of ATP–BeFx that proceeded more rapidly than in the presence of ATP alone and reached a twofold higher plateau than that achieved with AMP–PNP, the folding mediated by the α16mer that proceeded with much lower yields. A mutant of α16mer, trapα, which traps the unfolded and partially folded substrate protein, did not affect the ATP–BeFx-dependent GFP folding by β16mer but it suppressed that mediated by α16mer to the level of spontaneous folding. These results suggested that β16mer differed from the α16mer in nucleotide binding affinity or the rate of nucleotide hydrolysis. [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]

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]

Escherichia coli (recombinant cells) and λphage DNA in suspension liquid were exposed to pressure pulses of about 20 μs duration and amplitude of up to 14 MPa. These pulses were generated by a diaphragmless shock tube. The destruction of cells was monitored by the assay of phenylalanine dehydrogenase leaking from the recombinant cells and was found to increase remarkably at the peak pressure of higher than 12 MPa. A probability relation for the cell destruction expressed as a function of pressure was proposed. It is most likely that there exists a threshold pressure for the cell destruction. Fragmentation effects of shock waves on λphage DNA were analyzed by electrophoresis. They were enhanced by increasing the shock wave strength and the number of shots. Probability for the DNA fragmentation as a function of pressure and molecular size was estimated with HPLC. The larger size of the DNA was more easily fragmented. A threshold pressure does not seem to exist for the DNA fragmentation. [ABSTRACT FROM AUTHOR]

Leucine dehydrogenase ( l-leucine: NAD oxidoreductase, deaminating, EC 1.4.1.9) has been purified to homogeneity from a moderate thermophilic bacterium, Bacillus stearothermophilus. Am improved method of preparative slab gel electrophoresis was used effectively to purify it. The enzyme has a molecular mass of about 300,000 and consists of six subunits with identical molecular mass (Mr, 49,000). The enzyme does not lose its activity by heat treatment at 70° C for 20 min, and incubation in the pH range of 5.5-10.0 at 55° C for 5 min. It is stable in 10 mM phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol at over 1 month, and is resistant to detergent and ethanol treatment. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their keto analogs in the presence of NAD and NADH, respectively, as the coenzymes. The pH optima are 11 for the deamination of l-leucine, and 9.7 and 8.8 for the amination of α-ketoisocaproate and α-ketoisovalerate, respectively. The Michaelis constants were determined: 4.4 mM for l-leucine, 3.3 mM for l-valine, 1.4 mM for l-isoleucine and 0.49 mM for NAD in the oxidative deamination. The B. stearothermophilus enzyme shows similar catalytic properties, but higher activities than that from Bacillus sphaericus. [ABSTRACT FROM AUTHOR]

A correction to the article "Creation of a thermostable NADP+-dependent D-amino acid dehydrogenase from Ureibacillus thermosphaericus strain A1 meso-diaminopimelate dehydrogenase by site-directed mutagenesis," Hironaga Akita and colleagues in the July 13, 2012 issue is presented.

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]