Publications

• A novel trehalose-synthesizing glycosyltransferase from Pyrococcus horikoshii: molecular cloning and characterization.

  Ryu S.I., Park C.S., Cha J., Woo E.J., Lee S.B.

  A gene (ORF PH1035), annotated to encode an uncharacterized hypothetical protein in Pyrococcus horikoshii, was first cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by Ni-NTA affinity chromatography and its molecular mass was determined to be 49,871Da b ... >> More

  A gene (ORF PH1035), annotated to encode an uncharacterized hypothetical protein in Pyrococcus horikoshii, was first cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by Ni-NTA affinity chromatography and its molecular mass was determined to be 49,871Da by MALDI-TOF mass spectrometry. When the purified enzyme was reacted with nucleoside diphosphate-glucoses including UDP-glucose as a donor and glucose, rather than glucose-6-phosphate, as an acceptor, it specifically created a free trehalose. The enzyme was also able to partly hydrolyze the trehalose to glucose. The optimum pH was 5.5 and the enzyme was highly stable from pH 6 to 8. The deduced amino acid sequence showed a high homology with that of the glycosyl transferase group 1 (Pfam00534) in the BLAST search. The results suggest that the enzyme is a novel glycosyltransferase catalyzing the synthesis of the trehalose in the archaeon. << Less

  Biochem. Biophys. Res. Commun. 329:429-436(2005) [PubMed] [EuropePMC]

• Structural insights on the new mechanism of trehalose synthesis by trehalose synthase TreT from Pyrococcus horikoshii.

  Woo E.J., Ryu S.I., Song H.N., Jung T.Y., Yeon S.M., Lee H.A., Park B.C., Park K.H., Lee S.B.

  Many microorganisms produce trehalose for stability and survival against various environmental stresses. Unlike the widely distributed trehalose-biosynthetic pathway, which utilizes uridine diphosphate glucose and glucose-6-phosphate, the newly identified enzyme trehalose glycosyltransferring synt ... >> More

  Many microorganisms produce trehalose for stability and survival against various environmental stresses. Unlike the widely distributed trehalose-biosynthetic pathway, which utilizes uridine diphosphate glucose and glucose-6-phosphate, the newly identified enzyme trehalose glycosyltransferring synthase (TreT) from hyperthermophilic bacteria and archaea synthesizes an α,α-trehalose from nucleoside diphosphate glucose and glucose. In the present study, we determined the crystal structure of TreT from Pyrococcus horikoshii at 2.3 Å resolution to understand the detailed mechanism of this novel trehalose synthase. The conservation of essential residues in TreT and the high overall structural similarity of the N-terminal domain to that of trehalose phosphate synthase (TPS) imply that the catalytic reaction of TreT for trehalose synthesis would follow a similar mechanism to that of TPS. The acceptor binding site of TreT shows a wide and commodious groove and lacks the long flexible loop that plays a gating role in ligand binding in TPS. The observation of a wide space at the fissure between two domains and the relative shift of the N-domain in one of the crystal forms suggest that an interactive conformational change between two domains would occur, allowing a more compact architecture for catalysis. The structural analysis and biochemical data in this study provide a molecular basis for understanding the synthetic mechanism of trehalose, or the nucleotide sugar in reverse reaction of the TreT, in extremophiles that may have important industrial implications. << Less

  J Mol Biol 404:247-259(2010) [PubMed] [EuropePMC]

• A novel trehalose synthesizing pathway in the hyperthermophilic Crenarchaeon Thermoproteus tenax: the unidirectional TreT pathway.

  Kouril T., Zaparty M., Marrero J., Brinkmann H., Siebers B.

  In the genome of the hyperthermophilic archaeon Thermoproteus tenax a gene (treS/P) encoding a protein with similarity to annotated trehalose phosphorylase (TreP), trehalose synthase (TreS) and more recently characterized trehalose glycosyltransferring synthase (TreT) was identified. The treS/P ge ... >> More

  In the genome of the hyperthermophilic archaeon Thermoproteus tenax a gene (treS/P) encoding a protein with similarity to annotated trehalose phosphorylase (TreP), trehalose synthase (TreS) and more recently characterized trehalose glycosyltransferring synthase (TreT) was identified. The treS/P gene as well as an upstream located ORF of unknown function (orfY) were cloned, heterologously expressed in E. coli and purified. The enzymatic characterization of the putative TreS/P revealed TreT activity. However, contrary to the previously characterized reversible TreT from Thermococcus litoralis and Pyrococcus horikoshii, the T. tenax enzyme is unidirectional and catalyzes only the formation of trehalose from UDP (ADP)-glucose and glucose. The T. tenax enzyme differs from the reversible TreT of T. litoralis by its preference for UDP-glucose as co-substrate. Phylogenetic and comparative gene context analyses reveal a conserved organization of the unidirectional TreT and OrfY gene cluster that is present in many Archaea and a few Bacteria. In contrast, the reversible TreT pathway seems to be restricted to only a few archaeal (e.g. Thermococcales) and bacterial (Thermotogales) members. Here we present a new pathway exclusively involved in trehalose synthesis--the unidirectional TreT pathway--and discuss its physiological role as well as its phylogenetic distribution. << Less

  Arch Microbiol 190:355-369(2008) [PubMed] [EuropePMC]

• TreT, a novel trehalose glycosyltransferring synthase of the hyperthermophilic archaeon Thermococcus litoralis.

  Qu Q., Lee S.J., Boos W.

  The gene cluster in Thermococcus litoralis encoding a multicomponent and binding protein-dependent ABC transporter for trehalose and maltose contains an open reading frame of unknown function. We cloned this gene (now called treT), expressed it in Escherichia coli, purified the encoded protein, an ... >> More

  The gene cluster in Thermococcus litoralis encoding a multicomponent and binding protein-dependent ABC transporter for trehalose and maltose contains an open reading frame of unknown function. We cloned this gene (now called treT), expressed it in Escherichia coli, purified the encoded protein, and identified it as an enzyme forming trehalose and ADP from ADP-glucose and glucose. The enzyme can also use UDP- and GDP-glucose but with less efficiency. The reaction is reversible, and ADP-glucose plus glucose can also be formed from trehalose and ADP. The rate of reaction and the equilibrium favor the formation of trehalose. At 90 degrees C, the optimal temperature for the enzymatic reaction, the half-maximal concentration of ADP-glucose at saturating glucose concentrations is 1.14 mm and the V(max) is 160 units/mg protein. In the reverse reaction, the half-maximal concentration of trehalose at saturating ADP concentrations is 11.5 mm and the V(max) was estimated to be 17 units/mg protein. Under non-denaturating in vitro conditions the enzyme behaves as a dimer of identical subunits of 48 kDa. As the transporter encoded in the same gene cluster, TreT is induced by trehalose and maltose in the growth medium. << Less

  J. Biol. Chem. 279:47890-47897(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• A unique combination of genetic systems for the synthesis of trehalose in Rubrobacter xylanophilus: properties of a rare actinobacterial TreT.

  Nobre A., Alarico S., Fernandes C., Empadinhas N., da Costa M.S.

  Trehalose is the primary organic solute in Rubrobacter xylanophilus under all conditions tested, including those for optimal growth. We detected genes of four different pathways for trehalose synthesis in the genome of this organism, namely, the trehalose-6-phosphate synthase (Tps)/trehalose-6-pho ... >> More

  Trehalose is the primary organic solute in Rubrobacter xylanophilus under all conditions tested, including those for optimal growth. We detected genes of four different pathways for trehalose synthesis in the genome of this organism, namely, the trehalose-6-phosphate synthase (Tps)/trehalose-6-phosphate phosphatase (Tpp), TreS, TreY/TreZ, and TreT pathways. Moreover, R. xylanophilus is the only known member of the phylum Actinobacteria to harbor TreT. The Tps sequence is typically bacterial, but the Tpp sequence is closely related to eukaryotic counterparts. Both the Tps/Tpp and the TreT pathways were active in vivo, while the TreS and the TreY/TreZ pathways were not active under the growth conditions tested and appear not to contribute to the levels of trehalose observed. The genes from the active pathways were functionally expressed in Escherichia coli, and Tps was found to be highly specific for GDP-glucose, a rare feature among these enzymes. The trehalose-6-phosphate formed was specifically dephosphorylated to trehalose by Tpp. The recombinant TreT synthesized trehalose from different nucleoside diphosphate-glucose donors and glucose, but the activity in R. xylanophilus cell extracts was specific for ADP-glucose. The TreT could also catalyze trehalose hydrolysis in the presence of ADP, but with a very high K(m). Here, we functionally characterize two systems for the synthesis of trehalose in R. xylanophilus, a representative of an ancient lineage of the actinobacteria, and discuss a possible scenario for the exceptional occurrence of treT in this extremophilic bacterium. << Less

  J. Bacteriol. 190:7939-7946(2008) [PubMed] [EuropePMC]