Unusually broad substrate tolerance of a heat-stable archaeal sugar nucleotidyltransferase for the synthesis of sugar nucleotides.
Herein, we report the first cloning, recombinant expression, and synthetic utility of a sugar nucleotidyltransferase from any archaeal source and demonstrate by an electrospray ionization mass spectrometry (ESI-MS)-based assay its unusual tolerance of heat, pH, and sugar substrates. The metal-ion-dependent enzyme from Pyrococcus furiosus DSM 3638 showed a relatively high degree of acceptance of glucose-1-phosphate (Glc1P), mannose-1-phosphate (Man1P), galactose-1-phosphate (Gal1P), fucose-1-phosphate, glucosamine-1-phosphate, galactosamine-1-phosphate, and N-acetylglucosamine-1-phosphate with uridine and deoxythymidine triphosphate (UTP and dTTP, respectively). The apparent Michaelis constants for Glc1P, Man1P, and Gal1P are 13.0 +/- 0.7, 15 +/- 1, and 22 +/- 2 microM, respectively, with corresponding turnover numbers of 2.08, 1.65, and 1.32 s(-1), respectively. An initial velocity study indicated an ordered bi-bi catalytic mechanism for this enzyme. The temperature stability and inherently broad substrate tolerance of this archaeal enzyme promise an effective reagent for the rapid chemoenzymatic synthesis of a range of natural and unnatural sugar nucleotides for in vitro glycosylation studies and highlight the potential of archaea as a source of new enzymes for synthesis.
Mizanur
Rahman M
RM
Department of Chemistry and Plant Science Institute, Gilman Hall, Iowa State University, Ames, Iowa 50011-3111, USA.
Zea
Corbin J
CJ
Pohl
Nicola L
NL
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000096:biosynthesis*; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000737:chemistry / Q000378:metabolism*; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D009711:Nucleotides; D011994:Recombinant Proteins; D013403:Sugar Phosphates; D009713:Nucleotidyltransferases; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase