A novel trehalose-synthesizing glycosyltransferase from Pyrococcus horikoshii: molecular cloning and characterization.
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.
Ryu
Soo-In
SI
Research Institute of Food and Nutritional Sciences, Department of Food and Nutrition, Yonsei University, Seoul 120-749, Republic of Korea.
Park
Cheon-Seok
CS
Cha
Jaeho
J
Woo
Eui-Jeon
EJ
Lee
Soo-Bok
SB
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014199:Trehalose / Q000096:biosynthesis* / Q000737:chemistry*
D011994:Recombinant Proteins; D014199:Trehalose; D016695:Glycosyltransferases; D005947:Glucose