One-step synthesis of labeled sugar nucleotides for protein O-GlcNAc modification studies by chemical function analysis of an archaeal protein.
Herein we present the chemical function analysis of a recombinant sugar nucleotidyltransferase from the hyperthermophile Pyrococcus furiosus and its use in the one-pot synthesis of chloroacetyl- and alkyne-tagged analogues of uridinediphospho-N-acetylglucosamine (UDP-GlcNAc). The gene was originally annotated as a glucose-1-phosphate deoxythymidylyltransferase; however, kinetic analysis of a panel of sugar-1-phosphates with the protein shows that it is better described as a bifunctional protein that synthesizes UDP-GlcNAc from glucosamine-1-phosphate and acetyl coenzyme A (CoA). A new mass-spectrometry-based assay for the rapid analysis of the acyltransferase activity demonstrates that the enzyme can also accept cheaper truncated N-acetylcysteamine thioester substrates in place of the natural acetyl CoA. The enzyme can tolerate alkyne or chloride substitutions in the acyl moiety, thereby allowing the facile synthesis of tagged sugar nucleotides for future use in protein O-GlcNAc modification studies.
Mizanur
Rahman M
RM
Department of Chemistry, The Plant Sciences Institute, Gilman Hall, Iowa State University, Ames, Iowa 50011-3111, USA.
Jaipuri
Firoz A
FA
Pohl
Nicola L
NL
D019843:Archaeal Proteins / Q000378:metabolism*; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000378:metabolism*; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism*; D014530:Uridine Diphosphate / Q000378:metabolism
D019843:Archaeal Proteins; D009711:Nucleotides; D013403:Sugar Phosphates; D014530:Uridine Diphosphate; C098108:N-acetylglucopyranosylamine; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase; D005944:Glucosamine