Investigations of the Active Site of Saccharomyces cerevisiae Dolichyl-Phosphate-Mannose Synthase Using Fluorescent Labeled Dolichyl-Phosphate Derivatives

Abstract

Dolichol-phosphate mannose (Dol-P-Man) is a key mannosyl donor for the biosynthesis of N-linked oligosaccharides as well as for O-linked oligosaccharides on yeast glycoproteins, and for the synthesis of the glycosyl-phosphatidylinositol anchor found on many cell surface glycoproteins. It is synthesized by Dol-P-Man synthase which is the only glycosyltransferase in the dolichol pathway that has been expressed as an active protein, solubilized and purified in large enough quantities for structural investigations. Earlier studies showed that the enzyme is closely associated with membranes of endoplasmic reticulum with unique lipid requirements for its maximal activity. This potential target of antibiotic therapy is now being investigated at the molecular level to establish information about the structure of the enzyme as well as determine the nature and properties of the enzyme-phospholipid interactions. In this paper, we have determined the activities of the fluorescent labeled dolichyl-phosphate derivatives as well as the intramolecular distances between amino acid residues near the active site and/or the fluorophores of the substrate derivatives using fluorescence energy resonance transfer. These results also show that the conserved consensus sequence is not required by Dol-P-Man synthase neither for the recognition of Dol-P nor for the catalytic activity.

Novel prenyl-linked benzophenone substrate analogues of mycobacterial mannosyltransferases

“…In particular, we demonstrated that the transfer of Man from the mycobacterial C 50 / C 40 /C 35 [23]. In the present study, we reproduced these assays using mannosylated probes, generated via recombinant Mt-Ppm1/ D2, as the radiolabelled Man donor.…”

“…The results were very gratifying in relation to substrate specificity and photoinhibition. All ten probes were excellent substrates for the PPM synthase, illustrating that changes within the lipid portion [22], such as overall chain length, saturation, unsaturation, benzyl and cyclohexyl rings, are tolerated without a substantial decrease in activity [22,[35][36][37] (Table 1). Probe 7 yielded the best activity for the PPM synthase assay (Table 1), presumably representing an optimum in terms of hydrophobicity and solubility of the substrate.…”

“…In these studies, it has been shown that photoaffinity probes, based on naturally occurring prenols, are recognized by transferase enzymes and are taken up in much the same way as the natural substrates. Recently, fluorescent-based dolichyl phosphates modified at either internal β-, γ -or ω-isoprene units have been used as acceptors to examine the dolichyl phosphate Man synthase from Saccharomyces cerevisiae [35][36][37].…”

“…The biosynthetic relationship of PI → PIMs → LM → AraLAM has recently been supported by biochemical [17] and genetic studies [18][19][20][21]. Besra et al [17] demonstrated that Ac 1 PIM 2 (according to the nomenclature of Kordulakova et al [21]) is specifically extended by an addition of Manp residues from the alkali-stable sugar donor C 50 /C 40 /C 35 -PPM (where PPM stands for polyprenol monophosphomannose) to form higher PIMs and further to a 'linear' α(1 → 6)-LM via a PPM-dependent α(1 → 6)mannosyltransferase. The generation of PPM in M. tuberculosis H37Rv results from the transfer of Manp from GDP-Manp to polyprenyl phosphates catalysed by the C-terminal domain(s) of a PPM synthase (Mt-Ppm1), now termed as Mt-Ppm1/D2 [22].…”

Protein O-mannosylation: What we have learned from baker's yeast

“…Thereby Dpm1 can interact with both, the water-soluble mannosyl donor substrate GDP-Man in the cytosol and the highly hydrophobic mannosyl acceptor Dol-P within the ER membrane. Up to date, the three-dimensional (3D) crystal structure of yeast Dpm1 has not been resolved, however, usage of Dol-P and synthetic Dol-P analogs containing a fluorophore group provided a 3D structural model of the enzyme with bound GDP-Man, Dol-P and Mg 2+ ions to trace substrate binding and catalytic sites [36,37]. A two-domain structure was predicted with an N-terminal domain, which is made up of parallel ß-strands, and a C-terminal domain consisting of antiparallel ß-strands, each flanked by α-helices on both sides.…”