Unusually Broad Substrate Tolerance of a Heat-Stable Archaeal Sugar Nucleotidyltransferase for the Synthesis of Sugar Nucleotides

Abstract

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.

Biosynthetic Mechanism forL-Gulose in Main Polar Lipids ofThermoplasma acidophilumand Possible Resemblance to Plant Ascorbic Acid Biosynthesis

“…The two key enzymes in the characteristic non-phosphorylated Entner-Doudoroff pathway for Sulfolobus solfataricus have been reported by Lamble et al to be able to convert both glucose and galactose with almost the same activity as that for glucose/galactose. 51,52) This promiscuity in archaeal enzymes has been observed with glucose dehydrogenase in this organism, 53) as well as with nucleotidyltransferase 54) and glycogen synthase 55) in Pyrococcus. Our previous studies of calditol, a five-membered, carbocycle-containing lipid component of Sulfolobus, have strongly suggested the involvement of a putative calditol carbocycle cyclase with relatively low substrate specificity for glucose and galactose.…”

Characterization of Meiothermus taiwanensis Galactokinase and its Use in the One‐Pot Enzymatic Synthesis of Uridine Diphosphate‐Galactose and the Chemoenzymatic Synthesis of the Carbohydrate Antigen Stage Specific Embryonic Antigen‐3

“…In the preliminary enzymatic activity screening assay, the reaction of MtGalK was performed in a solution (100-200 mL) containing 8 mM Gal, 10 mM ATP, 5 mM MgCl 2 , 50 mM HEPES (pH 7.5), and 1 mg mL À1 purified MtGalK. The reaction was incubated at 37 8C for 0, 1, 2, 3, 4,5,6,7,8,9,10,15,20,25,30,40,50, 60, 90 and 120 min and was quenched by the addition of 200 mM EDTA to a final EDTA concentration of 20 mM. In a further optimal reaction condition analysis (temperature, metal ion cofactor and pH), the reaction was incubated for 5 min then quenched by the addition of EDTA.…”

Posttranslational Protein Modification inArchaea

“…Following transfer of the oligosaccharide moiety to a protein target, the phosphorylated dolichol carrier is recycled to its original topology. See references 247, 420, and 468, the text, and Table 3 resents the first analysis of an archaeal sugar nucleotidyltransferase (290). An N-acetylglucosamine transferase was also partially characterized from membranes of Halobacterium salinarum (281).…”