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Title:   Author  item
                                            XU, LIN - UNIV ROCHESTER MED CENTERitem
                                            Appell, Michaelitem
                                            KENNEDY, SCOTT - UNIV ROCHESTER MED CENTERitem
                                            Momany, Frankitem
                                            Price, Neil
  Submitted to: Biochemistry
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 8/20/2004
                                    Publication  Date: 10/26/2004
                                    Citation: Xu, L., Appell, M.D., Kennedy, S., Momany, F.A., Price, N.P. 2004. Conformational analysis of chirally-deuterated tunicamycin as an active site probe of UDP-N-acetylhexosamine:  polyprenol-p-N-acetylhexosamine-1-p translocases. Biochemistry. 43:13248-13255.Interpretive Summary: Antimicrobial compounds typically target essential functions in growing cells and produce their effects by disrupting these functions.  A family of enzymes called HexNAc-1-P translocases are important in the synthesis of a variety of microbial carbohydrates, particularly cell wall polysaccharides.  The compound tunicamycin targets these enzymes because of its structural similarity to the natural substrate and effectively blocks the reaction carried out by these enzymes.  In our research, we showed how the tunicamycin structurally interacts with its target, and the mechanisms of this interaction are described for the first time.  We also showed the binding of various divalent metal ions to tunicamycin, comparable to the binding that occurs in the translocase enzyme.  The work provides a valuable tool for researchers that will impact our understanding of how microbial polysaccharides are made.
                                Technical Abstract: 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 **1H- and **13C-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, mono-deuterated (S-6)-[**2H1]glucose has 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 **4C1 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 pseudo-galactopyranosyl 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 analog of the UDP-D-HexNAc substrate and is reversibly coordinated to the divalent metal cofactor in the D-HexNAc-1-P translocase active site.


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