Conformational analysis of chirally deuterated tunicamycin as an active site probe of UDP-N-acetylhexosamine:polyprenol-P N-acetylhexosamine-1-P translocases.
Tunicamycins are potent inhibitors of UDP-N-acetyl-D-hexosamine:polyprenol-phosphate N-acetylhexosamine-1-phosphate translocases (D-HexNAc-1-P translocases), a family of enzymes involved in bacterial cell wall synthesis and eukaryotic protein N-glycosylation. Structurally, tunicamycins consist of an 11-carbon dialdose core sugar called tunicamine that is N-linked at C-1' to uracil and O-linked at C-11' to N-acetylglucosamine (GlcNAc). The C-11' O-glycosidic linkage is highly unusual because it forms an alpha/beta anomeric-to-anomeric linkage to the 1-position of the GlcNAc residue. We have assigned the (1)H and (13)C NMR spectra of tunicamycin and have undertaken a conformational analysis from rotating angle nuclear Overhauser effect (ROESY) data. In addition, chirally deuterated tunicamycins produced by fermentation of Streptomyces chartreusis on chemically synthesized, monodeuterated (S-6)-[(2)H(1)]glucose have been used to assign the geminal H-6'a, H-6'b methylene bridge of the 11-carbon dialdose sugar, tunicamine. The tunicamine residue is shown to assume pseudo-D-ribofuranose and (4)C(1) pseudo-D-galactopyranosaminyl ring conformers. Conformation about the C-6' methylene bridge determines the relative orientation of these rings. The model predicts that tunicamycin forms a right-handed cupped structure, with the potential for divalent metal ion coordination at 5'-OH, 8'-OH, and the pseudogalactopyranosyl 7'-O ring oxygen. The formation of tunicamycin complexes with various divalent metal ions was confirmed experimentally by MALDI-TOF mass spectrometry. Our data support the hypothesis that tunicamycin is a structural analogue of the UDP-D-HexNAc substrate and is reversibly coordinated to the divalent metal cofactor in the D-HexNAc-1-P translocase active site.
Xu
Lin
L
University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, New York 14642, USA.
Appell
Michael
M
Kennedy
Scott
S
Momany
Frank A
FA
Price
Neil P J
NP
D001665:Binding Sites; D002236:Carbohydrate Conformation; D002413:Cations, Divalent / Q000378:metabolism; D041961:Deuterium Exchange Measurement / Q000379:methods; D004187:Disaccharides / Q000737:chemistry; D005688:Galactosamine / Q000031:analogs & derivatives* / Q000737:chemistry; D006027:Glycosides / Q000737:chemistry; D006838:Hydrocarbons; D007536:Isomerism; D008274:Magnesium / Q000378:metabolism; D008697:Methane / Q000031:analogs & derivatives* / Q000737:chemistry; D015335:Molecular Probes / Q000378:metabolism*; D017349:N-Acetylhexosaminyltransferases / Q000737:chemistry* / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity; D014415:Tunicamycin / Q000737:chemistry* / Q000378:metabolism*; D014498:Uracil / Q000737:chemistry
D002413:Cations, Divalent; D004187:Disaccharides; D006027:Glycosides; D006838:Hydrocarbons; D015335:Molecular Probes; D014415:Tunicamycin; C030011:carbene; D014498:Uracil; C076322:tunicamine; D005688:Galactosamine; D017349:N-Acetylhexosaminyltransferases; D008274:Magnesium; D008697:Methane