Fragment-based Screening of the Donor Substrate Specificity of Human Blood Group B Galactosyltransferase Using Saturation Transfer Difference NMR

Abstract

Saturation transfer difference NMR experiments on human blood group B ␣-(1,3)-galactosyltransferase (GTB) for the first time provide a comprehensive set of binding epitopes of donor substrate analogs in relation to the natural donor UDP-Gal. This study revealed that the enzyme binds several UDP-activated sugars, including UDP-Glc, UDP-GlcNAc, and UDP-GalNAc. In all cases, UDP is the dominant binding epitope. To identify the minimum requirements for specific binding, a detailed analysis utilizing a fragment-based approach was employed. The binding of donor substrate to GTB is essentially controlled by the base as a "molecular anchor." Uracil represents the smallest fragment that is recognized, whereas CDP, AMP, and GDP do not exhibit any significant binding affinity for the enzyme. The ribose and ␤-phosphate moieties increase the affinity of the ligands, whereas the pyranose sugar apparently weakens the binding, although this part of the molecule controls the specificity of the enzyme. Accordingly, UDP represents the best binder. The binding affinities of UDP-Gal, UDP-Glc, and UMP are about the same, but lower than that of UDP. Furthermore, we observed that ␤-D-galactose and ␣-D-galactose bind weakly to GTB. Whereas ␤-D-galactose binds to the acceptor and donor sites, it is suggested that ␣-D-galactose occupies a third hitherto unknown binding pocket. Finally, our experiments revealed that modulation of enzymatic activity by metal ions critically depends on the total enzyme concentration, raising the question as to which of the bivalent metal cations Mg 2؉ and Mn 2؉ is more relevant under physiological conditions.

WaaA of the Hyperthermophilic Bacterium Aquifex aeolicus Is a Monofunctional 3-Deoxy-d-manno-oct-2-ulosonic Acid Transferase Involved in Lipopolysaccharide Biosynthesis

“…However, our SPR measurements failed to detect any affinity of WaaA AAE for Kdo. It has been shown for other glycosyltransferases that the nucleotide and sugar moieties of the donor substrate play different roles in the interaction between the enzyme and the activated nucleotide sugar, with the nucleotide moiety being crucial to recognition of the substrate and the sugar being instead involved in determining the specificity of the enzyme (76). This supports the idea that CMP may guide CMP-Kdo to the donor substrate binding site of the Kdo transferase.…”

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

“…unproductive, because transfer to the H antigen acceptor does not readily occur (31,32). On the basis of NMR data for UDPGlc bound to GTB, Angulo et al (32) proposed a "tweezers" mechanism where Asp-302 and Glu-303 lock Gal in the tucked under conformation to facilitate formation of the transition state.…”

Enzymatic Basis for N-Glycan Sialylation

“…This interaction presumably makes a significant contribution to acceptor binding and likely accounts for the 80-fold increase in K m for lactose as an acceptor compared with N-acetyllactosamine (79). Binding interactions with the Neu5Ac residue of the sugar donor were less numerous, consistent with the primary binding affinity of sugar nucleotides to glycosyltransferases coming from the nucleotide component (80). Energetics of interaction with the nucleotide were not calculated, because this part of the molecule was restrained during the simulation.…”

“…Shown is the full MS spectra of the peptide LMNSQLVTTEKR that displays the observed mixture of a doubly charged unlabeled (major isotopic peak of 710.386 m/z) and labeled (major isotopic peak of 734.359 m/z) peptide that represents the difference between sulfur and selenium incorporation into the corresponding methionine. Note the unusual isotopic distribution of the labeled peptide due to the isotopic composition of selenium (a mixture of multiple isotopes where the abundance is 80 Se Ͼ 78 Se Ͼ 76 Se Ͼ 82 Se Ͼ 77 Se Ͼ 74 Se). Incorporation of selenium based on these peak areas is 79.3%, and when all Met-containing peptides were utilized for calculations, the incorporation was determined to be 73 Ϯ 8%.…”