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

Filipkowski, Paweł; Pietrow, Olga; Panek, Anna; Synowiecki, J.

doi:10.18388/abp.2012_2133

Cited by 17 publications

(17 citation statements)

References 38 publications

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“…In a number of GH13 enzymes, Tris has been shown to be a competitive inhibitor and is able to mimic a sugar molecule bound at the À1 subsite (Skov et al, 2002;Ravaud et al, 2007). An inhibitory effect of Tris on the activity of TS has also been observed (Filipkowski et al, 2012;Jiang et al, 2013). Our present assay results indicated that the activity of DrTS is inhibited strongly by Tris, with a IC 50 value of 4.88 mM in the presence of 50 mM maltose (Fig.…”

Section: The Substrate-binding Sitesupporting

confidence: 65%

Structures of trehalose synthase fromDeinococcus radioduransreveal that a closed conformation is involved in catalysis of the intramolecular isomerization

Wang¹,

Chow²,

Lin³

et al. 2014

Acta Crystallogr D Biol Crystallogr

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Trehalose synthase catalyzes the simple conversion of the inexpensive maltose into trehalose with a side reaction of hydrolysis. Here, the crystal structures of the wild type and the N253A mutant of Deinococcus radiodurans trehalose synthase (DrTS) in complex with the inhibitor Tris are reported. DrTS consists of a catalytic ( / ) 8 barrel, subdomain B, a C-terminal domain and two TS-unique subdomains (S7 and S8). The C-terminal domain and S8 contribute the majority of the dimeric interface. DrTS shares high structural homology with sucrose hydrolase, amylosucrase and sucrose isomerase in complex with sucrose, in particular a virtually identical activesite architecture and a similar substrate-induced rotation of subdomain B. The inhibitor Tris was bound and mimics a sugar at the À1 subsite. A maltose was modelled into the active site, and subsequent mutational analysis suggested that Tyr213, Glu320 and Glu324 are essential within the +1 subsite for the TS activity. In addition, the interaction networks between subdomains B and S7 seal the active-site entrance. Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 8-9-fold and an increased hydrolase activity by 1.5-1.8-fold. The N253A structure showed a small pore created for water entry. Therefore, our DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis.

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Section: The Substrate-binding Sitesupporting

confidence: 65%

Structures of trehalose synthase fromDeinococcus radioduransreveal that a closed conformation is involved in catalysis of the intramolecular isomerization

Wang¹,

Chow²,

Lin³

et al. 2014

Acta Crystallogr D Biol Crystallogr

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“…The deduced amino acid sequence of treS was used to perform a BLAST research of the NCBI and SwissProt databases. This search revealed that the protein has the highest similarity (84%) with trehalose synthase from Deinococcus radiodurans R1 [29]. As can be seen in Figure 1, multiple sequence alignments of this treS gene with 10 reported trehalose synthase revealed that they share several highly conserved amino acid motifs, such as SPLRDG/DGYDV/I, FPL/VMPRI/LF/Y, NHDELTLE, GIRRRLA/MPL, and so on.…”

Section: Resultsmentioning

confidence: 99%

“…With regard to these results, the recombinant TreS had a higher affinity to maltose and a favorite reaction direction toward the synthesis of trehalose. Interestingly, all reported TreS enzymes share the feature of a reversible conversion at different degrees [26,29]. …”

Section: Resultsmentioning

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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“…Nery et al [40] reported that an increase of trehalose content improves the radiation tolerance of microbial cells. Of the five pathways for trehalose biosynthesis, the maltose oligosaccharide synthase (TreY/ TreZ) pathway and the trehalose synthase (TreS) pathway exist in Deinococcus species [41][42][43]. Like other six closely related strains, genomic data predicted that strain SDU3-2 T also contained the two putative trehalose synthesis pathways, and the predicted related genes are shown in Table S5.…”

Section: Resistance and Associated Genesmentioning

confidence: 99%

Deinococcus terrestris sp. nov., a gamma ray- and ultraviolet-resistant bacterium isolated from soil

Wang

Chen

et al. 2020

International Journal of Systematic and Evolutionary Microbiology

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Strain SDU3-2T was isolated from a soil sample collected in Shandong Province, PR China. Cells of SDU3-2T were spherical, Gram-stain-positive, aerobic and non-motile. Cellular growth of the strain occurred at 25–45 °C, pH 5.5–8.5 and with 0–1.5 % (w/v) of NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain SDU3-2T was closest to the type strain Deinococcus murrayi ALT-1bT with a similarity of 95.2 %. The draft genome was 3.49 Mbp long with 69.2 mol% G+C content. Strain SDU3-2T exhibited high resistance to gamma radiation (D10 >12 kGy) and UV (D10 >900 J m−2). The strain encoded many genes for resistance to radiation and oxidative stress, which were highly conserved with other Deinococcus species, but possessed interspecific properties. The major fatty acids of SDU3-2T cells were C15 : 1 ω6c, C16 : 1 ω7c/C16 : 1 ω6c, and C17 : 1 ω8c, the major menaquinone was menaquinone-8, and the major polar lipids were an unidentified phosphoglycolipid, four unidentified glycolipids and an unidentified phospholipid. The average nucleotide identity and DNA–DNA hybridization results further indicated that strain SDU3-2T represents a new species in the genus Deinococcus , for which the name Deinococcus terrestris sp. nov. is proposed. The type strain is SDU3-2T (=CGMCC 1.17147T=KCTC 43098T).

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

Cited by 17 publications

References 38 publications

Structures of trehalose synthase fromDeinococcus radioduransreveal that a closed conformation is involved in catalysis of the intramolecular isomerization

Structures of trehalose synthase fromDeinococcus radioduransreveal that a closed conformation is involved in catalysis of the intramolecular isomerization

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

Deinococcus terrestris sp. nov., a gamma ray- and ultraviolet-resistant bacterium isolated from soil

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