A novel trehalose-synthesizing glycosyltransferase from Pyrococcus horikoshii

Molecular cloning and characterization

Soo In Ryu, Cheon Seok Park, Jaeho Cha, Eui Jeon Woo, Soo-Bok Lee

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

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 49,871 Da 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.

Original languageEnglish
Pages (from-to)429-436
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume329
Issue number2
DOIs
Publication statusPublished - 2005 Apr 8

Fingerprint

Pyrococcus horikoshii
Glycosyltransferases
Trehalose
Cloning
Molecular Cloning
Enzymes
Glucose
Uridine Diphosphate Glucose
Affinity chromatography
Glucose-6-Phosphate
Diphosphates
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Archaea
Molecular mass
Transferases
Affinity Chromatography
Nucleosides
Escherichia coli
Open Reading Frames
Mass spectrometry

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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abstract = "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 49,871 Da 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.",
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A novel trehalose-synthesizing glycosyltransferase from Pyrococcus horikoshii : Molecular cloning and characterization. / Ryu, Soo In; Park, Cheon Seok; Cha, Jaeho; Woo, Eui Jeon; Lee, Soo-Bok.

In: Biochemical and Biophysical Research Communications, Vol. 329, No. 2, 08.04.2005, p. 429-436.

Research output: Contribution to journalArticle

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