UDP_Glycosyltransferase

The UDP-glycosyltransferase UGT76G1 (PDB ID: 6INF) is a protein native to Stevia rebaudiana (S. rebaudiana) Bertoni, a shrub native to South America, and is capable of forming glycosidic linkages between glucose and various accepting substrates. UGT76G1 acts on steviol glycosidic substrates, including sugar molecules synthesized by members of the Stevia family (1,2). Steviol glycosides are a common zero-calorie sweetener that can be used as healthy alternatives to foods that are high in sugar. Since high-sugar diets are a major health concern, alternative sweeteners are important subjects of scientific study (1,3). Currently, the synthetic pathways for producing these steviol glycosides are inefficient processes. UGT76G1 catalyzes the β (1-3) addition of glucose onto stevioside to convert it to rebaudioside A. Stevioside has a bitter aftertaste, and the additional glucose makes a sweeter compound that is more desirable in consumer products (3). Furthermore, glycosylation and sugars play a key role in cell/cell signaling, and in protein synthesis, making the study of enzymes such as glycosyltransferases critical for further drug development to combat many diseases (4). UGT76G1 acts on a number of substrates with applications to the health and food industries, making this an important protein to study and understand.

Crystallization analysis of multiple different complexes with variants of UGT76G1 determined the structure of the protein. The complexes analyzed were wild type (WT) with uridine 5'-diphosphate (UDP), WT with UDP and rebaudioside A (Reb A), and WT with UDP and rubusoside (Rubu). Additionally, an H25A mutant of the protein was also crystallized with UDP. All the structures were within a root-mean-square-deviation of 0.4-0.9Å over all the α carbons, indicating that the spatial arrangement of all the complexes is essentially identical. The encoding region for UGT76G1 from S. rebaudiana was synthesized de novo with a C-terminal histidine tag for purification. The DNA was transformed into BL21 (DE3) Escherichia coli (E. coli) cells on the pET21a expression vector, which were used to express and purify UGT76G1. The purified protein mixed with 1mM UDP and the various steviol glucosides crystallized through the sitting-drop vapor-diffusion method at 20°C. The crystallization conditions were optimized, and the best crystals formed under 0.1M sodium citrate buffer at pH 5.4 and 20% PEG 4000. X-ray diffraction of the crystals collected data of crystals for each complex (1).

UGT76G1 exhibits a GT-B fold, characterized by two Rossman-like terminal domains with a β/α/β structure, that are loosely associated with an active site between the two domains (5). The N and C terminal domains both form parallel β-sheets. The C terminal domain consists of six β-strands, while the N terminal domain is made up of seven β-strands. The β-strands are connected by a combination of α-helices and loops. The entirety of the protein crystal structure has been determined in the PDB structure (1). The total molecular weight is 53.094 kDa, and the isoelectric point of the protein is pI 5.61 (6).

UGT76G1 is a single polypeptide chain of 466 residues that exists as a monomer in solution, and is 42% helical and 15% β-sheet (1,2). There are 17 β-strands made of 70 residues in the two parallel β-sheets, and there are 23 helices made of 199 residues in the structure (2). The folding of the protein follows the principle of hydrophobic collapse, with buried structures such as α-helices 3 and 4 comprised of mainly hydrophobic residues with a few polar side chains to coordinate with the substrate (1,2). The tertiary structure of the molecule is made of two main domains that associate at the active site (1). Since there is only one subunit chain, there is no quaternary structure associated with UGT76G1.

UGT76G1 serves to form glycosidic linkages between steviol substrates and glucose molecules. In effect, it adds a glucose to most viable steviosides. This occurs at the active site on the enzyme, which forms conserved substrate interactions that appear in other GT-B fold glycosyltransferases (1). The active site rests between the two terminal Rossman-like domains (5). In the active site, a molecule of uridine 5'-diphosphate (UDP) acts as the substrate, sitting on two alpha helices from the C terminal domain, and resting perpendicular to the C-terminal β-sheet (1). UDP is an ester of pyrophosphoric acid with the nucleoside uridine; it has a pyrophosphate group, a ribose, and the nucleobase uracil (7). The uracil ring participates in a π stacking interaction with the ring of Trp-338, and forms two hydrogen bonds with the backbone of Val-339. The ribose of UDP forms hydrogen bonds with Glu-364, and the O2 and O3 hydroxyls form a bidentate hydrogen bond to the amide of Asn-27. The α-phosphate oxygens of UDP interact electrostatically with the amides of Asn-360 and Ser-361. The β-phosphate of UDP interacts electrostatically with His-356 and Ser-283 (1).

In the reaction that forms the glycosidic linkage, UGT76G1 increases the nucleophilicity of the accepting glucose (the glucose on the stevioside), and positions it to attack the UDP-glucose donor (UDPG). Somewhat atypical for most sugar substrates in enzymes, the glucose on the nucleophile forms minimal hydrogen bonds with the protein. Instead, the interaction is dominated by hydrophobic interactions with the residues Leu-379, Phe-22, and Ile-90. There is one hydrogen bond that forms between the 3-hydroxyl oxygen in the nucleophilic glucose and the His-25 side chain. In the H25N mutant, all enzyme activity is eliminated, indicating that this interaction is crucial to the function of the glycosyltransferase (1).

The mechanism of glycosyltransferase catalysis appears to be highly conserved across all known enzymes. There are two possible stereochemical outcomes of glycosyltransferase catalysis; retention or inversion of the stereochemistry about the anomeric carbon (4). UGT76G1 inverts the stereochemistry of the substrate, indicating that UGT76G1 operates via an SN2 reaction (1,4). The nucleophile in the reaction is the hydroxyl on the accepting substrate (in this case, it is the stevioside), and the leaving group is the nucleotide on the donor (for UGT76G1, that would be the UDP that is left from the UDPG donor) (4). Though the mechanism for UGT76G1 is not fully understood, most glycosyltransferase SN2 reactions occur by deprotonation of the nucleophile to increase its nucleophilicity. Researchers have proposed that the His-25 residue of UGT76G1 acts to deprotonate the 3-hydroxyl of the stevioside substrate, priming it for nucleophilic attack of the UDPG donor (1). This appears to be consistent with the spatial arrangement of the crystal structures, and would explain why the H25N mutant is rendered incapable of function, despite the fact that the protein still folds properly (1,4).

Comparing the structure of UGT76G1 to proteins of similar sequence and function can help distinguish the conserved components of UGT76G1 from the portions of the structure that are less crucial to function. To this end, PSI-BLAST and Dali databases will identify proteins of similar primary and tertiary structures, respectively. The Dali server finds proteins with similar tertiary structures using a sum-of-pairs method that compares intramolecular distances. The similarity level in the Dali server is describe with a Z score; a Z score of 2 or higher indicates a significantly similar protein. PSI-BLAST searches for homology within primary structure to a given query. The amount of similarity between proteins is describe with an E value by PSI-BLAST. The E value is a report of the number of gaps that exist between the comparison proteins, the more gaps there are the greater the E value. An E value less than 0.05 is considered significant in protein structures.

A protein common to both searches (that is, one that exhibited similarity in both sequence and tertiary structure) was selected to compare to the structure of UGT76G1 (8,9). The selected protein was UDP-glucuronosyltransferase native to Homo sapiens (H. sapiens) (UGT2B7, PDB ID: 2O6L). The glucuronosyltransferase was chosen because it is native to H. sapiens, and may have some important and interesting connections to human processes. UGT2B7 from H. sapiens has an E value of 9e-08 in the PSI-BLAST search, and a Z score of 22.6 in the Dali server search (8,9). UGT2B7 has an MSE (L-peptide linking) ligand, rather than the UDP that is crystallized with UGT76G1 (10). Additionally, while UGT76G1 has two Rossman-like β/α/β domains at the termini, UGT2B7 has only one Rossman-like domain that is α/β/α. The UGT2B7 adopts a GT-B fold like UGT76G1, but while UGT76G1 catalyzes the addition of glucose molecules to a steviol substrate, UGT2B7 catalyzes the addition of glucuronic acid to a variety of substrates (11).

Overall, UGT76G1 is a protein with many important potential applications, from the food industry to drug development. Understanding the mechanism of glycosyltransferases can be applied to the synthesis of sweet molecules, but it can also play an important role in developing drugs for diseases that use glycosylation as biomolecular recognition tags. Future studies could focus on creating an efficient synthesis for sweeter, zero-calorie steviosides. Studies could also focus on ways to inhibit the UGT76G1 enzyme, which could prove useful in the study of certain diseases.