Substrate-induced conformational changes in glycosyltransferases

Background: Substrate hydrolysis has impeded structural investigation of human ABO(H) glycosyltransferase specificity. Results: Complexes with natural and isosteric non-hydrolyzable donor analogs show multiple stable intermediate donor binding conformations. Conclusion: Subtle stereochemical differences from natural donor prevent isosteric donor analog from displaying full mimicry. Significance: High resolution structural analysis provides insight into inhibitor development and the multistage process of substrate binding.

Section: Homologous Glycosyltransferases ␣-(133)-n-acetylgalactosaminmentioning

confidence: 99%

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner

Gagnon

Meloncelli

Zheng

et al. 2015

“…Interestingly, a similar model has been described for Gal/GalNAc specificity of ␤1,4-galactosyltransferases that distinguish vertebrates from invertebrates (31). Briefly, in these galactosyltransferases, one specific amino acid residue (residue 289) largely determines the enzyme specificity, and the amino acid has bulky side groups for Gal-transferases found among vertebrates, whereas the amino acid has small side groups for GalNAc-transferases of invertebrates (31)(32)(33).…”

Section: Discussionmentioning

confidence: 83%

Streptococcus pneumoniae Serotype 11D Has a Bispecific Glycosyltransferase and Expresses Two Different Capsular Polysaccharide Repeating Units

Oliver

Jones

Larson³

et al. 2013

Background: Streptococcus pneumoniae serotype 11D capsular polysaccharide (CPS) structure is unknown. Results: Serotype 11D PS contains two different repeating units; one has ␣GlcNAc, and the other contains ␣Glc. Conclusion: The 11D CPS is due to the bispecific glycosyltransferase WcrL. Based on codon 112, WcrL can transfer ␣Glc, ␣GlcNAc, or both. Significance: Minimal genetic changes can make bacteria produce different polysaccharides.

“…Mutating Tyr 289 to leucine, isoleucine, or asparagine made the Gal-transferase to a GalNAc-transferase (69). Furthermore, studies on several glycosyl transferases have shown that one or two flexible loops at the substrate binding site of the enzyme undergo marked conformational changes from an open to a closed conformation, in which the loop acts as a lid covering the bound donor (70). This flexibility may create a new acceptor-binding site or modulate it in combination with the conformational properties of the newly synthesized Man 2 GlcNAc 2 -PP-Dol oligosaccharide intermediate, thus altering Alg2 acceptor specificity.…”

Section: Discussionmentioning

confidence: 99%

Biochemical Characterization and Membrane Topology of Alg2 from Saccharomyces cerevisiae as a Bifunctional α1,3- and 1,6-Mannosyltransferase Involved in Lipid-linked Oligosaccharide Biosynthesis

Kämpf

Absmanner

Schwarz

et al. 2009

N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc 3 Man 9 GlcNAc 2 -PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the ␣1,3-and the ␣1,6-mannose-linked residue from GDP-mannose to Man 1 GlcNAc 2 -PP-Dol forming the Man 3 GlcNAc 2 -PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N-or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX 7 E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys 230 , as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.Asparagine-linked glycosylation is an essential protein modification highly conserved in eukaryotes (1-4), and several features of this pathway even occur in prokaryotes (5-7). In eukaryotes, biosynthesis of N-glycans starts with the assembly of the common core oligosaccharide precursor Glc 3 Man 9 GlcNAc 2 -PP-Dol, the glycan moiety of which is subsequently transferred onto selected Asn-Xaa-(Ser/Thr) acceptor sites of the nascent polypeptide chain by the oligosaccharyl-transferase complex (8 -10). The initial steps of the dolichol pathway up to Man 5 GlcNAc 2 -PP-Dol take place on the cytosolic site of the endoplasmic reticulum (ER), 2 using sugar nucleotides as glycosyl donors. Upon translocation of the heptasaccharide to the luminal site, which is facilitated by Rft1 (11) and another not yet identified protein (12), it is extended by four mannose and three glucose residues deriving from Man-P-Dol and Glc-P-Dol. It has been demonstrated that the pathway operates sequentially in an ordered fashion based on differences in the substrate specificity of the various glycosyltransferases (13). In the yeast Saccharomyces cerevisiae, alg mutants (for asparagine-linked glycosylation) have been isolated, defective in lipid-linked oligosaccharide (LLO) assembly (14 -17), and shown to be invaluable to define the pathway as well as to isolate the genes encoding the respective glycosyltransferases by complementing a particular phenotype characteristic of the respective mutant. Likewise various mutant cell lines from mammalian origin have been described that produce truncated lipid-linked oligosaccharides (18 -20).One of the tempe...