Structural Snapshots of β-1,4-Galactosyltransferase-I Along the Kinetic Pathway

During the catalytic cycle of β1,4-galactosyltransferase-1 (Gal-T1), upon the binding of Mn²⁺ followed by UDP–Gal, two flexible loops, a long and a short loop, change their conformation from open to closed. We have determined the crystal structures of a human M340H-Gal-T1 mutant in the open conformation (apo-enzyme), its Mn²⁺ and Mn²⁺–UDP–Gal-bound complexes, and of a pentenary complex of bovine Gal-T1–Mn²⁺–UDP–GalNAc-Glc–α-lactalbumin. These studies show that during the conformational changes in Gal-T1, the coordination of Mn²⁺ undergoes significant changes. It loses a coordination bond with a water molecule bound in the open conformation of Gal-T1 while forming a new coordination bond with another water molecule in the closed conformation, creating an active ground-state structure that facilitates enzyme catalysis. In the crystal structure of the pentenary complex, the N-acetylglucosamine (GlcNAc) moiety is found cleaved from UDP–GalNAc and is placed 2.7 Å away from the O4 oxygen atom of the acceptor Glc molecule, yet to form the product. The anomeric C1 atom of the cleaved GalNAc moiety has only two covalent bonds with its non-hydrogen atoms (O5 and C2 atoms), similar to either an oxocarbenium ion or N-acetylgalactal form, which are crystallographically indistinguishable at the present resolution. The structure also shows that the newly formed, metal-coordinating water molecule forms a hydrogen bond with the β-phosphate group of the cleaved UDP moiety. This hydrogen bond formation results in the rotation of the β-phosphate group of UDP away from the cleaved GalNAc moiety, thereby preventing the re-formation of the UDP–sugar during catalysis. Therefore, this water molecule plays an important role during catalysis in ensuring that the catalytic reaction proceeds in a forward direction.

Introduction

β1,4-Galactosyltransferase-1 (EC 2.4.1.38) (Gal-T1) is a Golgi resident type II membrane protein that catalyzes the transfer of galactose from UDP-Gal to N-acetylglucosamine (GlcNAc) in the presence of Mn²⁺, forming the product N-acetyllactosamine.1, 2, 3 In the presence of α-lactalbumin (α-LA) it transfers galactose to glucose (Glc) to form lactose. Among the many glycosyltransferases known to date, the kinetic mechanism of Gal-T1 has been extensively studied. It follows an ordered sequential mechanism: first Mn²⁺ binds to the enzyme, followed by UDP–Gal, and then the acceptor substrate, GlcNAc4, 5 (Figure 1). These studies have further shown that the binding of the metal ion to the enzyme is essential for the binding of UDP–Gal. After the transfer of galactose to the acceptor, the disaccharide product is ejected, and UDP is released. Recent crystallographic studies on the catalytic domain of this enzyme from a bovine source, residues 134–402, with various substrate complexes, have shown that the conformational changes that the enzyme undergoes upon Mn²⁺–UDP–Gal binding involves two flexible regions: a short loop, residues 313–316 (309–312 in human Gal-T1), and a long loop, residues 345 to 365 (341–361 in human Gal-T1).6, 7, 8, 9 Based on these conformational changes, the enzyme kinetic mechanism can be completely described (Figure 1). These studies have further provided the details on the binding of both the donor and acceptor substrates and their influence on the conformational changes.

More recently we have shown that the metal binding site located at the N terminus hinge region of the long flexible loop influences the conformational dynamics of the loop.⁹ We found that when Met344, the metal binding residue of the enzyme, was mutated to His, although the affinity for the Mn²⁺ and the donor substrate increased, the enzyme could not efficiently revert to the open conformation essential for efficient catalysis.⁹ Interestingly, when the Mg²⁺ was used as the metal ion cofactor that binds weakly to the mutant enzyme, the flexible loops went back and forth between the open and closed conformation easily, increasing the overall efficiency of the catalytic cycle. Thus, for Gal-T1 to go through the conformational changes more efficiently, it is essential that at position 344, either Met residue has to engage Mn²⁺ or His residue has to engage Mg²⁺ during the catalytic cycle of the enzyme.⁹ We have exploited this property to successfully crystallize the His mutant enzyme from bovine or human sources, in the presence of Mn²⁺ and UDP–hexanolamine, with various oligosaccharide accepter substrates, which is otherwise not possible with the wild-type enzyme.9, 10

In order to better understand the role of the metal ion, it is essential to have the metal ion-bound structures of the enzyme, both in the open and closed conformations, and also in the presence and absence of substrates. So far, the binding of Mn²⁺ to the enzyme is known only in the closed conformation in the presence of UDP or UDP–Gal6, 7, 8, 9, 10. An attempt made by Gastinel et al.¹¹ to observe its binding to the wild-type enzyme in the open conformation by soaking crystals with UDP–Gal and Mn²⁺ failed to locate any Mn²⁺. Therefore, we chose to investigate the binding of the Mn²⁺ to the mutant enzyme, Met344His–Gal-T1, which has been shown to bind to Mn²⁺ with a strength nearly 15-fold greater than that of the wild-type.⁹ We present results of the crystallographic investigations on the apo-enzyme, Mn²⁺–enzyme and Mn²⁺–UDP–Gal–enzyme complexes in the open conformation. We compare the metal coordination in these crystal structures with the previously determined structure of the same mutant of bovine Gal-T1, b-M344H–Gal-T1, crystallized in the closed conformation in complex with Mn²⁺ and UDP–Gal.⁹ In relation to metal ion binding, we have also determined the crystal structure of the mutant b-M344H–Gal-T1 in the closed conformation in the presence of mouse α-LA, in complex with Ca²⁺ and UDP–Gal.

Finally, to explore the enzyme's catalytic mechanism, we crystallized the b-Gal-T1 enzyme in the presence of Mn²⁺ and UDP–GalNAc, a less preferred sugar donor with which it has only 0.1% activity compared to UDP–Gal, and with the acceptor glucose (Glc) in complex with α-LA. In the crystal structure of the pentenary complex, we found that GalNAc moiety is cleaved from UDP–GalNAc and displaced towards the bound acceptor Glc. Due to the steric hindrance caused by the Tyr289 residue,¹² the cleaved GalNAc was unable to form the disaccharide product, but exists with the anomeric C1 atom having only two covalent bonds with non-hydrogen atoms O5 and C2. This crystal structure suggests a possible role of a conserved water molecule in the catalytic pocket of both inverting and retaining glycosyltransferases and general catalytic mechanism for all the inverting glycosyltransferases.