Novel UDP-GalNAc Derivative Structures Provide Insight into the Donor Specificity of Human Blood Group Glycosyltransferase

Abstract

Two closely related glycosyltransferases are responsible for the final step of the biosynthesis of ABO(H) human blood group A and B antigens. The two enzymes differ by only four amino acid residues, which determine whether the enzymes transfer GalNAc from UDP-GalNAc or Gal from UDP-Gal to the H-antigen acceptor. The enzymes belong to the class of GT-A folded enzymes, grouped as GT6 in the CAZy database, and are characterized by a single domain with a metal dependent retaining reaction mechanism. However, the exact role of the four amino acid residues in the specificity of the enzymes is still unresolved. In this study, we report the first structural information of a dual specificity cis-AB blood group glycosyltransferase in complex with a synthetic UDP-GalNAc derivative. Interestingly, the GalNAc moiety adopts an unusual yet catalytically productive conformation in the binding pocket, which is different from the “tucked under” conformation previously observed for the UDP-Gal donor. In addition, we show that this UDP-GalNAc derivative in complex with the H-antigen acceptor provokes the same unusual binding pocket closure as seen for the corresponding UDP-Gal derivative. Despite this, the two derivatives show vastly different kinetic properties. Our results provide a important structural insight into the donor substrate specificity and utilization in blood group biosynthesis, which can very likely be exploited for the development of new glycosyltransferase inhibitors and probes.

Structure and Mechanism of Staphylococcus aureus TarS, the Wall Teichoic Acid β-glycosyltransferase Involved in Methicillin Resistance

“…Departure of the leaving UDP is stabilized by the coordinating Mn 2+ and leads to glycosyl transfer with inversion of the stereochemistry of the GlcNAc anomeric centre. UDP-GlcNAc itself is furthermore observed to assume a “tucked under” conformation (as common in other glycosyltransferases [38,48–55]), where the GlcNAc sugar is tucked below the plane of the UDP diphosphates, allowing exposure of the scissile bond to nucleophilic attack (Fig 4B). In TarS, D178, situated 6.2 Å away on the β-face of the C1 anomeric carbon, is suitably positioned to act as a Brønsted base catalyst for the incoming acceptor (Fig 4B), and shows good spatial agreement with the catalytic aspartate of SpsA [56], a prototypical inverting GTA from the GT2 family (sharing 28% identity with TarS), when the structures are superimposed (overall main chain rmsd of 1.1 Å over 107 atom pairs) (S6A Fig).…”

Emerging structural insights into glycosyltransferase-mediated synthesis of glycans

“…The result is an inversion of the anomeric configuration of the product 5,9 . In most instances the enzyme-bound sugar nucleotide donor assumes a 'folded back' conformation during sugar transfer, in contrast to the more extended conformation of the donor in solution [16][17][18] . Significant conformational change in loop regions of the enzyme may also occur upon binding the sugar donor 13 .…”

Nucleoside Diphosphate Sugar Analogues that Target Glycosyltransferases

“…More-recents tudies on two closely related GTst hat are responsible for the final step of the biosynthesis of ABO human blood group Aa nd Ba ntigens revealed thatc ompounds 67 a and 67 d interfere with the folding of an internal loop and the Cterminus, which is essential for catalysis. [66] Additional specificity studies on those GTsw ere performed by analyzing the structure of ac omplexo fA A(gly)B and compound 67 d. [67] 5-Substituted UDPÀGal analogues (67)c ould also be prepared enzymatically by using d-glucose-1-phosphate uridyltransferase (EC 2.2.7.9), followed by the action of d-Gal epimerase (EC 5.1.3.2). However,i ng eneral, low yields (5-23 %) were obtained and the conversion of the gluco compounds into the corresponding galacto derivatives was the problematic step.…”