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

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• 作者：Rahman M. Mizanur ; Corbin J. Zea ; and Nicola L. Pohl
• 刊名：Journal of the American Chemical Society
• 出版年：2004
• 出版时间：December 15, 2004
• 年：2004
• 卷：126
• 期：49
• 页码：15993 - 15998
• 全文大小：114K
• 年卷期：v.126,no.49(December 15, 2004)
• ISSN：1520-5126

文摘

Herein, we report the first cloning, recombinant expression, and synthetic utility of a sugarnucleotidyltransferase from any archaeal source and demonstrate by an electrospray ionization massspectrometry (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 acceptanceof 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-phosphatewith uridine and deoxythymidine triphosphate (UTP and dTTP, respectively). The apparent Michaelisconstants for Glc1P, Man1P, and Gal1P are 13.0 ± 0.7, 15 ± 1, and 22 ± 2 ges/entities/mgr.gif">M, respectively, withcorresponding turnover numbers of 2.08, 1.65, and 1.32 s^-1, respectively. An initial velocity study indicatedan ordered bi-bi catalytic mechanism for this enzyme. The temperature stability and inherently broadsubstrate tolerance of this archaeal enzyme promise an effective reagent for the rapid chemoenzymaticsynthesis of a range of natural and unnatural sugar nucleotides for in vitro glycosylation studies and highlightthe potential of archaea as a source of new enzymes for synthesis.