ABO(H) Blood Group A and B Glycosyltransferases Recognize Substrate via Specific Conformational Changes

Yu Bai,Svetlana N Borisova,Mattias Persson,Todd L Lowary,Nina O.L Seto,Monica M Palcic,James A Letts,Javier A Alfaro,Stephen V Evans,Robert Polakowski,Ruixiang Blake Zheng

doi:10.1074/jbc.m708669200

Abstract

The final step in the enzymatic synthesis of the ABO(H) blood group A and B antigens is catalyzed by two closely related glycosyltransferases, an alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and an alpha-(1-->3)-galactosyltransferase (GTB). Of their 354 amino acid residues, GTA and GTB differ by only four "critical" residues. High resolution structures for GTB and the GTA/GTB chimeric enzymes GTB/G176R and GTB/G176R/G235S bound to a panel of donor and acceptor analog substrates reveal "open," "semi-closed," and "closed" conformations as the enzymes go from the unliganded to the liganded states. In the open form the internal polypeptide loop (amino acid residues 177-195) adjacent to the active site in the unliganded or H antigen-bound enzymes is composed of two alpha-helices spanning Arg(180)-Met(186) and Arg(188)-Asp(194), respectively. The semi-closed and closed forms of the enzymes are generated by binding of UDP or of UDP and H antigen analogs, respectively, and show that these helices merge to form a single distorted helical structure with alternating alpha-3(10)-alpha character that partially occludes the active site. The closed form is distinguished from the semi-closed form by the ordering of the final nine C-terminal residues through the formation of hydrogen bonds to both UDP and H antigen analogs. The semi-closed forms for various mutants generally show significantly more disorder than the open forms, whereas the closed forms display little or no disorder depending strongly on the identity of residue 176. Finally, the use of synthetic analogs reveals how H antigen acceptor binding can be critical in stabilizing the closed conformation. These structures demonstrate a delicately balanced substrate recognition mechanism and give insight on critical aspects of donor and acceptor specificity, on the order of substrate binding, and on the requirements for catalysis.

Highlights

Glycosyltransferases synthesize carbohydrate moieties of glycoconjugates by catalyzing the sequential addition of monosaccharides from specific donors to specific acceptors
We report the kinetic characterization of several chimeric enzymes along with high resolution structures of GTB (BBBB) and the chimeric enzyme ABBB in their unliganded states, BBBB and ABBB in the presence of UDP, BBBB and AABB in the presence of synthetic H antigen disaccharide ␣-L-Fucp-(132)-␤-D-GalpO(CH2)7CH3, BBBB and ABBB in the presence of UDP, and the H antigen acceptor analog ␣-L-2-deoxy-Fucp-(132)-␤-D-3-aminoGalp-O(CH2)7CH3, BBBB, and ABBB and in the presence of both UDP and H antigen disaccharide, and AABB in complex with UDP-Gal and the H antigen acceptor analog ␣-L-Fucp-(132)-␤-D-3-deoxy-Galp-O(CH2)7CH3
In the 12 structures determined, BBBB, ABBB, and AABB crystals were soaked with combinations of UDP, UDP-Gal, H antigen disaccharide (HA), 3-deoxy-Gal-H antigen disaccharide, and 2-deoxy-Fuc-3-amino-Gal-H antigen disaccharide

Summary

Introduction

Glycosyltransferases synthesize carbohydrate moieties of glycoconjugates by catalyzing the sequential addition of monosaccharides from specific donors to specific acceptors. Structural studies have revealed that specific internal sections of polypeptide adjacent to the active site are often observed to be flexible or completely disordered These internal loops have been suggested to restrict water access to the active site, as well as act in donor recognition and catalysis [3], including the inverting enzymes ␤4Gal-T1 [12], GnT-I [13], GlcAT-I [14], and GlcAT-P [15]; the retaining enzymes EXTL2 [16] ␣-(133)GalT [17, 18], GTA, and GTB [19, 20]; the microbial inverting SpsA [21] and CstII [22]; and the retaining microbial enzyme LgtC [23]. The retaining ␣-(133)-galactosyltransferase (␣-(133)GalT) is the enzyme most homologous to GTA/GTB in sequence and structure, and it has been reported to display substrate-induced conformational changes [18]. Subsequent studies have shown that part of the disorder of the internal loop was because of the presence of a heavy atom, and that crystals of the mutant enzyme GTB/C209A grown in the absence of heavy atoms display a smaller disordered segment of the internal loop consisting of residues 177–187 [20]

Results

Discussion

Conclusion