Structural Insights on the New Mechanism of Trehalose Synthesis by Trehalose Synthase TreT from Pyrococcus horikoshii

Woo, Eui-Jeon; Ryu, Soo In; Song, Ho Taek; Jung, Tae‐Yang; Yeon, Sei Mee; Lee, Hyun Ah; Park, Byoung Chul; Park, Kwan Hwa; Lee, Soo Bok

doi:10.1016/j.jmb.2010.09.056

Cited by 23 publications

(28 citation statements)

References 34 publications

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“…Indeed, a conformational change of ca. 4 Å has been observed between the sugar donor and acceptor binding domains for the protein crystal structure of TreT from Pyrococcus horikoshii (PDB accession number 2X6Q), which closes upon substrate binding (PDB accession number 2XMP), highlighting the importance of conformational flexibility (23). Moreover, conformational flexibility upon substrate binding has also been observed for trehalose phosphate synthase (OtsA) from E. coli K-12 (66), glycogen synthase (58), and ␣-fucosyltransferase V (59).…”

Section: Discussionmentioning

confidence: 99%

“…Several metabolic pathways for the biosynthesis of trehalose have been found in nature ( Fig. 1) and include the following: (i) trehalose synthase (TreS) interconverting maltose to trehalose (9); (ii) maltooligosyltrehalose synthase (TreYZ) hydrolyzing maltodextrins to trehalose (10,11); (iii) inverting trehalose phosphorylase (TreP inv ) (12)(13)(14)(15) adding ␣-D-glucose-1-phosphate or (iv) retaining trehalose phosphorylase (TreP ret ) (16)(17)(18)(19) adding ␤-D-glucose-1-phosphate to glucose, producing trehalose and phosphate; (v) trehalose transferase (TreT) using D-glucose and a nucleotide diphosphate (NDP) sugar to produce D-trehalose (20)(21)(22)(23)(24)(25); (vi) trehalose phosphate synthase (OtsA) producing D-trehalose-6-phosphate from D-glucose-6-phosphate and a nucleotide sugar (26)(27)(28)(29)(30). In contrast to trehalose phosphate synthase, the LeLoir glycosyltransferase TreT does not require the use of additional 6-phosphate (OtsA), avoiding sequential dephosphorylation of the nonreducing disaccharides, and therefore is of particular interest for industrial food applications (31,32).…”

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confidence: 99%

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Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Mestrom

Marsden

Dieters

et al. 2019

Appl Environ Microbiol

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LeLoir glycosyltransferases are important biocatalysts for the production of glycosidic bonds in natural products, chiral building blocks, and pharmaceuticals. Trehalose transferase (TreT) is of particular interest since it catalyzes the stereo-and enantioselective ␣,␣-(1¡1) coupling of a nucleotide sugar donor and monosaccharide acceptor for the synthesis of disaccharide derivatives. Heterologously expressed thermophilic trehalose transferases were found to be intrinsically aggregation prone and are mainly expressed as catalytically active inclusion bodies in Escherichia coli. To disfavor protein aggregation, the thermostable protein mCherry was explored as a fluorescent protein tag. The fusion of mCherry to trehalose transferase from Pyrobaculum yellowstonensis (PyTreT) demonstrated increased protein solubility. Chaotropic agents like guanidine or the divalent cations Mn(II), Ca(II), and Mg(II) enhanced the enzyme activity of the fusion protein. The thermodynamic equilibrium constant, K eq , for the reversible synthesis of trehalose from glucose and a nucleotide sugar was determined in both the synthesis and hydrolysis directions utilizing UDPglucose and ADP-glucose, respectively. UDP-glucose was shown to achieve higher conversions than ADP-glucose, highlighting the importance of the choice of nucleotide sugars for LeLoir glycosyltransferases under thermodynamic control. IMPORTANCE The heterologous expression of proteins in Escherichia coli is of great relevance for their functional and structural characterization and applications. However, the formation of insoluble inclusion bodies is observed in approximately 70% of all cases, and the subsequent effects can range from reduced soluble protein yields to a complete failure of the expression system. Here, we present an efficient methodology for the production and analysis of a thermostable, aggregation-prone trehalose transferase (TreT) from Pyrobaculum yellowstonensis via its fusion with mCherry as a thermostable fluorescent protein tag. This fusion strategy allowed for increased enzyme stability and solubility and could be applied to other (thermostable) proteins, allowing rapid visualization and quantification of the mCherry-fused protein of interest. Finally, we have demonstrated that the enzymatic synthesis of trehalose from glucose and a nucleotide sugar is reversible by approaching the thermodynamic equilibrium in both the synthesis and hydrolysis directions. Our results show that uridine establishes an equilibrium constant which is more in favor of the product trehalose than when adenosine is employed as the nucleotide under identical conditions. The influence of different nucleotides on the reaction can be generalized for all LeLoir glycosyltransferases under thermodynamic control as the position of the equilibrium depends solely on the reaction conditions and is not affected by the nature of the catalyst.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Mestrom

Marsden

Dieters

et al. 2019

Appl Environ Microbiol

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“…The phosphate is then removed by trehalose-phosphate phosphatase (TPP, EC 3.1.3.12) (OtsB in E. coli ) to give free trehalose [30]. The second pathway involves a trehalose glycosyltransferring synthase (TreT, EC 2.4.1.245) that catalyzes the synthesis of trehalose using nucleoside diphosphate glucose (NDPG), such as UDPG, as a donor and glucose as an acceptor [31]. The third pathway utilizes trehalose phosphorylase (TreP, EC 2.4.1.64) to catalyze a reversible reaction in which it hydrolyzes trehalose in the presence of inorganic phosphate to form glucose-1-phosphate and glucose and, inversely, gives rise to trehalose from both products in vitro [32].…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of a Novel Trehalose Synthase Gene Derived from Saline-Alkali Soil Metagenomes

et al. 2013

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A novel trehalose synthase (TreS) gene was identified from a metagenomic library of saline-alkali soil by a simple activity-based screening system. Sequence analysis revealed that TreS encodes a protein of 552 amino acids, with a deduced molecular weight of 63.3 kDa. After being overexpressed in Escherichia coli and purified, the enzymatic properties of TreS were investigated. The recombinant TreS displayed its optimal activity at pH 9.0 and 45 °C, and the addition of most common metal ions (1 or 30 mM) had no inhibition effect on the enzymatic activity evidently, except for the divalent metal ions Zn2+ and Hg2+. Kinetic analysis showed that the recombinant TreS had a 4.1-fold higher catalytic efficientcy (Kcat/K m) for maltose than for trehalose. The maximum conversion rate of maltose into trehalose by the TreS was reached more than 78% at a relatively high maltose concentration (30%), making it a good candidate in the large-scale production of trehalsoe after further study. In addition, five amino acid residues, His172, Asp201, Glu251, His318 and Asp319, were shown to be conserved in the TreS, which were also important for glycosyl hydrolase family 13 enzyme catalysis.

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“…Enzymes involved in different pathways of trehalose biosynthesis were extensively studied (Schiraldi et al, 2002;Woo et al, 2010). There are known microorganisms which have only a single pathway, but some of them have two and even three trehalose biosynthesis pathways (Avonce, 2006).…”

Section: Introductionmentioning

confidence: 99%

Properties of recombinant trehalose synthase from Deinococcus radiodurans expressed in Escherichia coli.

Filipkowski

Pietrow²,

Panek³

et al. 2012

Acta Biochim Pol

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A trehalose synthase gene from Deinococcus radiodurans (DSMZ 20539) containing 1659 bp reading frame encoding 552 amino acids was amplified using PCR. The gene was finally ligated into pET30Ek/LIC vector and expressed after isopropyl β-d-thiogalactopyranoside induction in Escherichia coli (DE3) Rosetta pLysS. The recombinant trehalose synthase (DraTreS) containing a His 6 -tag at the C-terminus was purified by metal affinity chromatography and characterized. The expressed enzyme is a homodimer with molecular mass of 126.9 kDa and exhibits the highest activity of 11.35 U/mg at pH 7.6 and at 30oC. DraTreS activity was almost unchanged after 2 h preincubation at 45oC and pH 7.6, and retained about 56% of maximal value after 8 h incubation at 50oC. The DraTreS was strongly inhibited by Cu 2+ , Hg 2+ , Zn 2+ , Al 3+ and 10 mM Tris. The K m value of maltose conversion was 290.7 mM.

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Structural Insights on the New Mechanism of Trehalose Synthesis by Trehalose Synthase TreT from Pyrococcus horikoshii

Cited by 23 publications

References 34 publications

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Identification and Characterization of a Novel Trehalose Synthase Gene Derived from Saline-Alkali Soil Metagenomes

Properties of recombinant trehalose synthase from Deinococcus radiodurans expressed in Escherichia coli.

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