Structure of a metal-independent bacterial glycosyltransferase that catalyzes the synthesis of histo-blood group A antigen

Abstract

Histo-blood group antigens (HBGAs) are a source of antigenic variation between individuals that modulates resistance and susceptibility to pathogens and is a barrier to the spread of enveloped viruses. HBGAs are also produced by a few prokaryotes where they are synthesized by glycosyltransferases (GTs) related to human HBGA synthases. Here we report the first structure of a bacterial GT of this family, from an intestinal resident, Bacteroides ovatus. Unlike its mammalian homologues and other GTs with similar folds, this protein lacks a metal-binding Asp-X-Asp motif and is fully active in the absence of divalent metal ions, yet is strikingly similar in structure and in its interactions with substrates to structurally characterized mammalian metal-dependent mammalian homologues. This shows how an apparently major divergence in catalytic properties can be accommodated by minor structural adjustments and illustrates the structural underpinnings of horizontal transfer of a functional gene from prokaryotes to vertebrates.

Structures of Complexes of a Metal-independent Glycosyltransferase GT6 from Bacteroides ovatus with UDP-N-Acetylgalactosamine (UDP-GalNAc) and Its Hydrolysis Products

“…Previous isothermal titration calorimetry studies have shown that this mutation has little net effect on the Δ G for UDP-GalNAc binding (both about −5.8 kcal/mol), but this reflects mutually compensating effects on the Δ H and T Δ S of binding (changes from −23.5 to −16.4 kcal/mol and 17.7 to 10.6 kcal/mol, respectively), suggesting that the mutation may weaken noncovalent interactions and reduce substrate-induced conformational rearrangements (13). Structures A, C, and B are models for complexes in the hydrolysis reaction catalyzed by BoGT6a, -C, and -B, being earlier and later steps in product release.…”

Thermodynamic Signature of Substrates and Substrate Analogs Binding to Human Blood Group B Galactosyltransferase from Isothermal Titration Calorimetry Experiments

“…In that case though, affinity for the acceptor substrate LacNAc has been too low to be measured by ITC. Recently, a retaining bacterial glycosyltransferase (BoGT6a) that catalyzes the formation of A‐antigen has been described . The enzyme has a surprising structural similarity with GTA and belongs to the same glycosyltransferase family, but it functions in the absence of bivalent metal cations.…”

“…These studies have shown that binding of donor and acceptor substrates is mutually enhanced. An increase of acceptor substrate affinity in the presence of donor substrate has also been reported for bovine α‐(1,3)‐galactosyltransferase (α3GT), and lately for a bacterial galactosyltransferase (BoGT6a) from B. ovatus that is structurally closely related to GTB, human blood group A N ‐acetylgalactosaminyltransferase (GTA) and α3GT but lacks a metal ion binding pocket . All these enzymes display a so‐called GT‐A fold and belong to glycosyltransferase family 6 (GT6) suggesting similar mechanistic behavior.…”

“…Such data are ideally obtained from isothermal titration calorimetry (ITC) but due to limited amounts of material or solubility problems such experiments are often difficult to perform. Therefore, ITC data on the binding of substrates to glycosyltransferases are sparse . Other methods such as surface plasmon resonance (SPR), NMR titrations, and most recently electrospray ionization mass spectrometry (ESI‐MS) delivered binding affinities but do not provide direct access to binding enthalpies.…”

“…Binding of acceptor substrates lactose and 2′‐fucosyllactose to α3GT and BoGT6a is too weak and outside the measuring range of ITC. Only in the presence of UDP binding enthalpies and dissociation constants have been obtained, again showing an unfavorable entropic contribution to binding . For GTB and the closely related GTA a van't Hoff analysis of temperature dependent affinity data from ESI‐MS has shown that on binding of H‐disaccharide acceptor to GTB or GTA, there is only very little (unfavorable) change in entropy .…”