Structure-Function of Galactosyltransferase sub-family members and Design of Novel Glycosyltransferases: Glycosyltransferases, a superfamily of enzymes, are involved in the synthesis of carbohydrate moieties of glycoproteins, glycolipids and glycosaminoglycans. The oligosaccharide moieties perform a variety of specific cellular functions during cell growth and cell-cell interactions, cell adhesion and fertilization, in the modulation of growth factor receptors, immune defense, inflammation, and viral as well as parasitic infections. Profound changes occur in the oligosaccharide structures during cellular development, differentiation, and tumorigenesis and in many disease states. Defective glycan synthesis has been shown to have serious pathological consequences and result in several human diseases. Glycosyltransferases synthesize these oligosaccharides by transferring a monosaccharide moiety of an activated sugar donor to the acceptor molecule. Most of the enzymes require a metal ion cofactor. The majority of these enzymes in the eukaryotic cell are anchored in the Golgi compartment, as type II membrane proteins. They have a short N-terminal cytoplasmic domain, a membrane-spanning region, as well as a stem and a C-terminal catalytic domain that face the Golgi-lumen. The reaction catalyzed by these enzymes follows a kinetic mechanism in which the metal ion and sugar-nucleotide bind first to the enzyme, followed by the acceptor. After the glycosyl moiety of the sugar-nucleotide donor is transferred to the acceptor, the saccharide product is ejected out, the release of the nucleotide and the metal ion follows to return the enzyme to its original state for a new round of catalytic cycle. The sugar transfer occurs either with the retention or inversion of the configuration at the anomeric carbon atom of the sugar donor. X-ray crystal structures of the catalytic domain of many glycosyltransferases, including beta-1,4-galactosylransferase (b4Gal-T1), free or complexed with substrates, have revealed that upon binding the donor substrate, one or two flexible loops undergo a marked conformational change, from an open to a closed conformation, simultaneously creating an acceptor binding site. Thus, the loop acts as a lid covering the bound donor substrate. After completion of the transfer of the glycosyl unit to the acceptor, the saccharide product is ejected; the loop reverts to its native conformation to release the remaining nucleotide moiety. The specificity of the sugar donor is determined by a few residues in the sugar-nucleotide binding pocket of the enzyme, which are conserved among the family members from different species. Crystallographic studies on the bovine b4Gal-T1 have shown that the primary metal binding site is located at the hinge region of a long flexible loop, which upon Mn2+ and UDP-Gal binding changes from an open to a closed conformation. This conformational change creates an oligosaccharide binding site in the enzyme. Neither UDP nor UDP analogues efficiently induce these conformational changes in the wild-type enzyme, thereby restricting the structural analysis of the acceptor binding site. The binding of Mn2+ involves an uncommon coordination to the S atom of Met344 residue; when it is mutated to His, the mutant M344H, in the presence of Mn2+ and UDP-hexanolamine, readily changes to closed conformation, facilitating the structural analysis of the enzyme bound with an oligosaccharide acceptor. Although the mutant M344H loses 98% of its Mn2+ dependent activity, it exhibits 25% of its activity in the presence of Mg2+ion. The enzyme kinetic studies with the mutant Met344His-Gal-T1 have shown that when Mn2+ is used during the catalytic cycle, at the product release phase the mutant enzyme is unable to return to the open conformation to release UDP and Mn2+. Conversely, when Mg2+ is used, due to its lower affinity, the mutant returns to the open conformation easily, indicating that the conformational dynamics of the long flexible loop are influenced by the metal ion binding. The crystal structures of M344H-Gal-T1 in complex with either UDP-Gal-Mn2+ or UDP-Gal-Mg2+, determined at 2.3 resolutions, show that the mutant enzyme in these complexes is in closed conformation, and the coordination stereochemistry of Mg2+ is quite similar to that of Mn2+ion. Although either Mn2+ or Mg2+, together with UDP-Gal, bind and change the conformation of the M344H mutant to the closed one, it is the Mg2+ complex that engages efficiently in catalyses. Thus, this property enabled us to crystallize the M344H mutant for the first time with the acceptor substrate chitobiose and other oligosaccharides (see project Z01 BC 010042) in the presence of UDP-hexanolamine and Mn2+. The crystal structure solved at 2.3 resolution reveals that the GlcNAc residue at the nonreducing end of chitobiose makes extensive hydrophobic interactions with the highly conserved Tyr286 residue. We have determined the crystal structure of Met344His mutant of human b4Gal-T1, M344H-Gal-T1 in an apo-form and bound with Mn2+ and bound with Mn2+-UDP-Gal to get the snapshots of the catalytic cycle of the enzyme. These crystal structures show Mn2+ binding to the enzyme in an open conformation, organization of the water molecules and freezing the hinge region residues of the flexible loop, followed by UDP-Gal binding and closed conformation of the flexible loop. Furthermore, in order to capture the transition state complex in the crystal structure, we have exploited the very poor N-acetylgalactosaminyltransferase activity (GalNAc-T) of the b4Gal-T1 in the presence of LA towards Glc. We have determined the crystal structure of b4Gal-T1- LA complex in the presence of UDP-GalNAc, Mn2+ and Glc at 2.0 resolutions. In the crystal structure both the donor and acceptor substrates are clearly observed. However, the GalNAc moiety of the donor is hydrolyzed from the UDP-GalNAc and found 3.0 from the P(beta) phosphate oxygen atom, displaced towards the acceptor, Glc molecule. The anomeric C1 atom of the GalNAc moiety is flat, existing in 4H3 conformation, an oxocarbenium-ion-like transition state, with only two covalent bonds with the non-hydrogen atoms O5 and C2. The steric hindrance caused by Tyr286, the residue corresponding to bovine Tyr289, acts as a molecular brake on the GalNAc moiety, the hydrolyzed GalNAc is trapped in its oxocarbenium-ion-like state and is placed 2.8 away from the O4 oxygen atom of the Glc molecule, yet to form the product GalNAcb1-4Glc. The trapped oxocarbenium-ion-like state has confirmed the catalytic mechanism proposed for the inverting glycosyltransferases, such as b1,4-Gal-T1. With the detailed structural information already obtained, the design of novel glycosyltransferases with broader or requisite donor and acceptor specificities has been possible. Investigating the structure and function of human b4Gal-T family members: We are currently expanding our biochemical and structural investigations to six human b4Gal-T1 family members, b4Gal-T1 to b4Gal-T7. Each member of the sub-family is differentially expressed in various tissues. All seven members of the sub-family have exclusive specificity to the donor substrate UDP-Gal and each transfers Gal in a beta1-4 linkage to a specific acceptor sugar. Among the family members, b4Gal-T5 and b4Gal-T6 are highly homologous (with a sequence identity of 95%), and they both show least sequence identity with the catalytic domain of b4Gal-T1 (32% and 30% for b4Gal-T5 and b4Gal-T6, respectively).
