Journal of Molecular Biology
Structural Snapshots of β-1,4-Galactosyltransferase-I Along the Kinetic Pathway
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.
Section snippets
Overall structure of the h-M340H–Gal-T1 mutant in the present crystal structures
The mutant h-M340H–Gal-T1 crystallized in an open conformation in the absence of the metal ion and substrates. In the apo-enzyme, residues 343 to 353 could not be located (Figure 2(a)). In the structure of the Mn2+-bound complex, however, because of the presence of the bulky hydrated metal ion at the N-terminal hinge region of the long flexible loop, three more residues, 343 to 345, have been located. In the Mn2+-UDP–Gal-bound complex in the open conformation, only the residues 345 to 353 could
Construction of the catalytic domain of the bovine Gal-T1 double mutant, b-Gal-T1–W312C-P401C
To increase the in vitro folding efficiency of the catalytic domain of bovine recombinant Gal-T1 (b-Gal-T1), we mutated the residues Trp312 to Cys312 and Pro401 to Cys401 for the following reasons.
The catalytic domain of Gal-T1 and its mutants are expressed in Escherichia coli as inclusion bodies.30 These proteins fold in vitro with about 12% folding efficiency. The crystal structure of b-Gal-T1 shows that at the N-terminal region, residue Cys134 forms a disulfide bond with Cys176, while at the
Acknowledgements
We thank Dr Alexander Wlodawer and Dr Xinhua Ji of the Molecular Crystallography Laboratory, NCI, and Professor Soma Kumar of Georgetown University for the critical reading of the manuscript. We thank Dr Zbigniew Dauter, NCI, Brookhaven National Laboratory, for his help in data collection. We also thank Professor S. S. Withers, University of British Columbia for his valuable comments and suggestions. This research was supported by the Intramural Research Program of the NIH, National Cancer
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