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

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 ␣-(133)-N-acetylgalactosaminyltransferase (GTA) and an ␣-(133)-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 Glycosyltransferases synthesize carbohydrate moieties of glycoconjugates by catalyzing the sequential addition of monosaccharides from specific donors to specific acceptors. The ubiquitous presence of glycolipids and glycoproteins in all living systems underlines the importance of the glycosyltransferases superfamily, and the DNA of all domains of life encode for a large number of these enzymes (1). To date, crystal structures of glycosyltransferases have displayed a high degree of structural similarity even when there is low sequence homology (2-4). As such, glycosyltransferases provide an excellent example of the preferential conservation of structural phenotype over the conservation of sequence identity (2), which indicates that the mechanism of glycosylation, although not yet fully understood, has been conserved.

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

“…For example, AAAA refers to GTA, BBBB refers to GTB, and AABB refers to the chimera with the first two critical residues of GTA and the last two critical residues of GTB. Previous findings implicate the first amino acid in enzyme turnover (12,26), the second and third in acceptor recognition (14,27), and the third and fourth in donor selection (14,17,25,28,29).…”

“…In the absence of substrate, these enzymes tend to adopt the "open" conformation, where both regions have higher disorder and/or the internal loop faces away from the active site (12). When UDP and manganese ion (Mn 2ϩ ) bind, there is a shift to the "semi-closed" conformation, where the internal loop is more ordered and oriented toward UDP so as to occlude the active site entrance, whereas the C-terminal loop remains disordered (12). With the addition of UDP or UDP-Gal in combination with acceptor or acceptor analog, GTA and GTB are observed to assume the "closed" state associated with catalytic activity (Fig.…”

“…1B): an internal loop (amino acids 176 -188) and C-terminal ␣-helix (amino acids 346 -354) (12,14). In the absence of substrate, these enzymes tend to adopt the "open" conformation, where both regions have higher disorder and/or the internal loop faces away from the active site (12).…”

“…These enzymes are essential for normal cell development, and aberrant GT function can result in a number of infections and inflammatory disease states (1). Despite their functional and physiological diversity, GTs exhibit structural phenotype conservation even in the absence of sequence homology (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). There are currently 97 GT families based on sequence identity (2,13), with almost all of the enzymes falling into two major fold types: GT-A and GT-B.…”

“…1B): an internal loop (amino acids 176 -188) and C-terminal ␣-helix (amino acids 346 -354) (12,14). In the absence of substrate, these enzymes tend to adopt the "open" conformation, where both regions have higher disorder and/or the internal loop faces away from the active site (12). When UDP and manganese ion (Mn 2ϩ ) bind, there is a shift to the "semi-closed" conformation, where the internal loop is more ordered and oriented toward UDP so as to occlude the active site entrance, whereas the C-terminal loop remains disordered (12).…”