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The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by coexpression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and nonsubstrate amino acids such as l -Arg and l -Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea. [ABSTRACT FROM AUTHOR]

The orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH. L-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thaliana AK-HseDH. However, the concentration required to inhibit the activity was much lower (K0.5 = 37 μM) than that needed to inhibit the A. thaliana enzyme (K0.5 = 500 μM). In contrast to A. thaliana AK-HseDH, Hse oxidation of the T. maritima enzyme was almost impervious to inhibition by L-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritima AK-HseDH is likely responsible for the unique sensitivity to L-threonine. Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK-HseDH: bifunctional aspartate kinase-homoserine dehydrogenase; AsaDH: aspartate-β-semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T). [ABSTRACT FROM AUTHOR]

It is believed that the plastids in green plants lost peptidoglycan (i.e. a bacterial cell wall-containing d -amino acids) during their evolution from an endosymbiotic cyanobacterium. Although wall-like structures could not be detected in the plastids of green plants, the moss Physcomitrella patens has the genes required to generate peptidoglycan (Mur genes), and knocking out these genes causes defects in chloroplast division. Here, we generated P. patens knockout lines (∆Pp- ddl) for a homolog of the bacterial peptidoglycan-synthetic gene encoding d -Ala: d -Ala ligase. ∆Pp- ddl had a macrochloroplast phenotype, similar to other Mur knockout lines. The addition of d -Ala- d -Ala (DA-DA) to the medium suppressed the appearance of giant chloroplasts in ∆Pp- ddl , but the addition of l -Ala- l -Ala (LA-LA), DA-LA, LA-DA, or d -Ala did not. Recently, a metabolic method for labeling bacterial peptidoglycan was established using ethynyl-DA-DA (EDA-DA) and click chemistry to attach an azide-modified fluorophore to the ethynyl group. The ∆Pp- ddl line complemented with EDA-DA showed that moss chloroplasts are completely surrounded by peptidoglycan. Our findings strongly suggest that the moss plastids have a peptidoglycan wall containing d -amino acids. By contrast, no plastid phenotypes were observed in the T-DNA tagged ddl mutant lines of Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

The article focuses on a study related to the evaluation of enzymes activity isolated from hyperthermophile Pyrobaculum islandicum. As per the study, the enzymes isolated from the species have various application in bioprocess engineering. The study highlights the molecular mechanism behind the stability of unboiled Nicotinamide Adenine Dinucleotide (NAD) dependent glutamate dehydrogenase. The study further concludes that the enzyme is extremely thermostable.

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.

Like an alpinist continuously seeking virgin peaks to climb, Kenji Soda has investigated a variety of unique enzymes for which there was little or no information available; and by doing so he opened up a variety of new fields in enzyme science and technology. In particular, he has promoted the study of enzymes requiring vitamin B-derived cofactors such as FAD, NAD(P) and pyridoxal 5′-phosphate, shedding light on their reaction mechanisms, enzymological properties, crystal structures and potential practical applications. Highlighted in this review are the studies of enzymes acting on d-amino acids and sulphur/selenium-containing amino acids and those from thermophilic and psychrophilic bacteria. [ABSTRACT FROM PUBLISHER]

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]

The gene encoding the ATP-dependent glucokinase of hyperthermophilic archaeon Aeropyrum pernix was identified, cloned, and functionally expressed in Escherichia coli. The deduced amino acid sequence showed 40% identity to that of the putative glucokinase from hyperthermophilic archaeon Pyrobacurum aerophilum. The purified recombinant enzyme was a monomer with a molecular mass of 35 kDa. The enzyme retained its full activity on heating at 70°C for 10 min and retained 65% of the activity after 10-min incubation at 100°C. The enzyme exclusively catalyzed the phosphorylation of d-glucose using ATP as a phosphoryl donor. ITP was accepted in addition to ATP. The rate dependence with both glucose and ATP followed Michaelis-Menten kinetics, with apparent Km values of 0.054 and 0.50 mM, respectively. The enzyme activity required divalent cations; Mg2+, which was most effective, could partially be replaced by Mn2+ or Ca2+. Phylogenetic analysis revealed that the glucokinase from A. pernix does not belong to the clusters of enzymes found in bacteria and eukarya. This is the first description of the characteristics of an ATP-dependent glucokinase from an archaeon. [ABSTRACT FROM PUBLISHER]

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]

The article focuses on a study which examines the effect of orally administering L-Serum-L-Tyrosine (SY) dipeptide on the brain of a serine deficiency disease model mouse to attain the efficient delivery of L-Tyrosine and L-Serum into the mouse brain. The study concluded that SY administration was more effective than L-Tyr administration at increasing the L-Tyr level in the brain and serum.

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.