Structure-Function of Galactosyltransferase sub-family members and Design of Novel Glycosyltransferases: Defective glycan synthesis has been shown to have serious pathological consequences and result in several human diseases.
Our aim has been to determine the structure and function of galactosyl-transferase (Gal-T) family members which, in the presence of a metal ion, transfer galactose (Gal) from UDP-Gal to an oligosaccharide acceptor and generate a disaccharide unit containing galactose in the complex oligosaccharide structures of glycoconjugates. A member of beta-1,4-galactosylransferase sub-family, b4Gal-T1, in the presence of substrates, interacts with alpha-lactalbumin (LA), a mammary gland specific protein, and forms lactose synthase (LS) complex that alters the acceptor specificity of the enzyme towards glucose (Glc) to produce lactose. The study on these proteins has been facilitated due to our successful in vitro folding of these proteins from inclusion bodies produced in E. coli, and we have shown that these proteins require oxido-shuffling agents and the stem region attached to the catalytic domains of galactosyltransferases that enhances their in vitro folding efficiency several fold. The structural and biochemical investigations on b4Gal-T1 and LS have revealed that they are akin to an exquisite mechanical device with two well-coordinated flexible loops that are contained within the b4Gal-T1 catalytic domain. The smaller one has a Trp residue (Trp314) flanked by glycine residues. The larger one comprises amino acid residues 345 to 365. Upon substrate binding, the Trp314 side chain moves to lock the sugar nucleotide in the binding site, while the large loop undergoes a conformational change, masking the sugar nucleotide binding site, and creates the oligosaccharide binding cavity which is also a protein-protein interacting site for the partner LA, in the LS complex. The limited proteolysis and the crystal structure studies with the Trp314Ala mutant shows the crucial role Trp314 plays in the conformational state of the long loop, in the binding of substrates and in the catalytic mechanism of the enzyme. The primary metal binding site is located at the hinge region of the long flexible loop that upon conformational change positions His347 in a way that it coordinates with Mn2+. Furthermore, Mn2+ at this site coordinates with Asp254 of D252VD254 sequence, with two oxygen atoms of UDP, and forms an uncommon coordination to the Sd atom of Met344 residue. The sixth coordination is with a water molecule, thus forming an octahedral metal ion coordination. Asp254 and His347 are strong metal binding ligands, while Met344 coordinates less strongly and can be substituted by serine, glutamine or alanine. However, when Met344 is substituted with His the mutant exhibits enzymatic activity in the presence of alkaline earth metals, which do not activate the wild-type enzyme. In the presence of Mg2+, the mutant exhibits 25% of the catalytic activity observed with the wild-type enzyme in the presence of Mn2+. It also has higher Km for the substrates. The crystal structures of M344H-Gal-T1 in complex with either UDP-GalMn2+ or UDP-GalMg2+, determined at 2.3 resolutions, show that the coordination stereochemistry of Mg2+ is quite similar to that of Mn2+. The His344 mutant exhibits stronger coordination bond with a metal ion compared to Met344 in the wild-type enzyme and that results in reduced kcat by not only interfering with the ability of the long flexible loop to undergo the required conformational changes during the catalytic cycle but also by interfering with the formation of the transition state complex. The mutant M344H crystallizes in the closed conformation in the presence of UDP-hexanolamine and Mn2+ and thus enabled us to co-crystallize several oligosaccharide acceptors. Only in conformation II do b4Gal-T1 and LA form the LS complex, enabling b4Gal-T1 to choose the new substrate glucose. The interaction of LA with b4Gal-T1 in conformation II also stabilizes the sugar-nucleotide-enzyme complex, kinetically enhancing the sugar transfer, even from the less preferred sugar nucleotides. Structure based design of novel glycosyltransferases: The information derived from the crystal structures of ?b4Gal-T1 with less preferred donor or acceptor substrates has been used to design the mutants of ?b4Gal-T1 that have preferences for different donor and acceptor substrates and has allowed us to synthesize oligosaccharides that are otherwise chemically difficult to synthesize. Previously, based on the structural information of the b4Gal-T1-UDP-GalNAc-LA complex, we have mutated Tyr289 to Leu289 and thereby made b4Gal-T1 to transfer Gal or GalNAc from their sugar nucleitides with equal effeciency. Now the crystal structure of Gal-T1-LA-UDP-Glc complex reveals that O4 hydroxyl group of Glc moiety forms a hydrogen bond with the side chain carboxylate group of Glu317, catalytically important residue of b4Gal-T1, and this may be responsible for the low Glc-T activity exhibited by Gal-T1. We have mutated Arg228 which forms the base of the catalytic pocket, where its positively charged guanidine group is placed between the side chain carboxylate group of Asp252 and Glu317 residues. In order to keep the positive charge between these two negatively charged side chain carboxylate groups, and still form a hydrogen bond with the side chain of Glu317, Arg228 was replaced by Lysine. As expected, the single mutant R228K exhibits enhanced Glc-T activity compared to wt-Gal-T1and LA further enhances this activity nearly 6 fold more.
