Aglycone Substrates Research Articles

Solvent Engineering for Nonpolar Substrate Glycosylation Catalyzed by the UDP-Glucose-Dependent Glycosyltransferase UGT71E5: Intensification of the Synthesis of 15-Hydroxy Cinmethylin β-d-Glucoside.

Sugar nucleotide-dependent glycosyltransferases are powerful catalysts of the glycosylation of natural products and xenobiotics. The low solubility of the aglycone substrate often limits the synthetic efficiency of the transformation catalyzed. Here, we explored different approaches of solvent engineering for reaction intensification of β-glycosylation of 15HCM (a C15-hydroxylated, plant detoxification metabolite of the herbicide cinmethylin) catalyzed by safflower UGT71E5 using UDP-glucose as the donor substrate. Use of a cosolvent (DMSO, ethanol, and acetonitrile; ≤50 vol %) or a water-immiscible solvent (n-dodecane, n-heptane, n-hexane, and 1-hexene) was ineffective due to enzyme activity and stability, both impaired ≥10-fold compared to a pure aqueous solvent. Complexation in 2-hydroxypropyl-β-cyclodextrin enabled dissolution of 50 mM 15HCM while retaining the UGT71E5 activity (∼0.32 U/mg) and stability. Using UDP-glucose recycling, 15HCM was converted completely, and 15HCM β-d-glucoside was isolated in 90% yield (∼150 mg). Collectively, this study highlights the requirement for a mild, enzyme-compatible strategy for aglycone solubility enhancement in glycosyltransferase catalysis applied to glycoside synthesis.

Point Mutations at a Key Site Alter the Cytochrome P450 OleP Structural Dynamics.

Substrate binding to the cytochrome P450 OleP is coupled to a large open-to-closed transition that remodels the active site, minimizing its exposure to the external solvent. When the aglycone substrate binds, a small empty cavity is formed between the I and G helices, the BC loop, and the substrate itself, where solvent molecules accumulate mediating substrate-enzyme interactions. Herein, we analyzed the role of this cavity in substrate binding to OleP by producing three mutants (E89Y, G92W, and S240Y) to decrease its volume. The crystal structures of the OleP mutants in the closed state bound to the aglycone 6DEB showed that G92W and S240Y occupied the cavity, providing additional contact points with the substrate. Conversely, mutation E89Y induces a flipped-out conformation of this amino acid side chain, that points towards the bulk, increasing the empty volume. Equilibrium titrations and molecular dynamic simulations indicate that the presence of a bulky residue within the cavity impacts the binding properties of the enzyme, perturbing the conformational space explored by the complexes. Our data highlight the relevance of this region in OleP substrate binding and suggest that it represents a key substrate-protein contact site to consider in the perspective of redirecting its activity towards alternative compounds.

UGT76B1 controls the growth-immunity trade-off during systemic acquired resistance

UGT76B1 controls the growth-immunity trade-off during systemic acquired resistance

Glycorandomization: A promising diversification strategy for the drug development.

Glycorandomization is a natural product derivatization strategy in which different sugar moieties are linked to the aglycone part of the naturally existing glycosides to create glycorandomized libraries. Sugars attached to the natural products are responsible for affecting their solubility, mechanism of action, target recognition, and toxicity and thus, by changing the sugar part, these properties could be modified. Glycorandomization can be done via two approaches (i) a synthetic approach known as neoglycorandomization, and (ii) chemoenzymatic approach including in-vitro and in-vivo glycorandomization. Glycorandomization can be a promising technology for the drug discovery that has proved its potential to improve pharmacokinetic (solubility) and pharmacodynamic profile (mechanism of action, toxicity, and target recognition) of the parent compounds. The substrate flexibility of glycosyltransferases and other enzymes towards sugars and/or aglycone substrates has made this technique versatile. Further, the enzymes can be altered by genetic engineering to generate glycorandomized libraries of diverse natural product scaffolds. This technique has the potential to produce new compounds that can be helpful to the mankind by treating the threatening disease states. This review covers the different strategies for glycorandomization as a tool in drug discovery and development. The fundamentals of glycorandomization, different types, and further development of differentially glycorandomized libraries of natural products and small molecule based drugs have been discussed.

Lifetimes of the Aglycone Substrates of Specifier Proteins, the Autonomous Iron Enzymes That Dictate the Products of the Glucosinolate-Myrosinase Defense System in Brassica Plants.

Specifier proteins (SPs) are components of the glucosinolate-myrosinase defense system found in plants of the order Brassicales (brassicas). Glucosinolates (GLSs) comprise at least 150 known S-(β-d-glucopyranosyl)thiohydroximate-O-sulfonate compounds, each with a distinguishing side chain linked to the central carbon. Following tissue injury, the enzyme myrosinase (MYR) promiscuously hydrolyzes the common thioglycosidic linkage of GLSs to produce unstable aglycone intermediates, which can readily undergo a Lossen-like rearrangement to the corresponding organoisothiocyanates. The known SPs share a common protein architecture but redirect the breakdown of aglycones to different stable products: epithionitrile (ESP), nitrile (NSP), or thiocyanate (TFP). The different effects of these products on brassica consumers motivate efforts to understand the defense response in chemical detail. Experimental analysis of SP mechanisms is challenged by the instability of the aglycones and would be facilitated by knowledge of their lifetimes. We developed a spectrophotometric method that we used to monitor the rearrangement reactions of the MYR-generated aglycones from nine GLSs, discovering that their half-lives (t1/2) vary by a factor of more than 50, from <3 to 150 s (22 °C). The t1/2 of the sinigrin-derived allyl aglycone (34 s), which can form the epithionitrile product (1-cyano-2,3-epithiopropane) in the presence of ESP, proved to be sufficient to enable spatial and temporal separation of the MYR and ESP reactions. The results confirm recent proposals that ESP is an autonomous iron-dependent enzyme that intercepts the unstable aglycone rather than a direct effector of MYR. Knowledge of aglycone lifetimes will enable elucidation of how the various SPs reroute aglycones to different products.

Short-chain dehydrogenase/reductase governs steroidal specialized metabolites structural diversity and toxicity in the genus Solanum

Thousands of specialized, steroidal metabolites are found in a wide spectrum of plants. These include the steroidal glycoalkaloids (SGAs), produced primarily by most species of the genus Solanum, and metabolites belonging to the steroidal saponins class that are widespread throughout the plant kingdom. SGAs play a protective role in plants and have potent activity in mammals, including antinutritional effects in humans. The presence or absence of the double bond at the C-5,6 position (unsaturated and saturated, respectively) creates vast structural diversity within this metabolite class and determines the degree of SGA toxicity. For many years, the elimination of the double bond from unsaturated SGAs was presumed to occur through a single hydrogenation step. In contrast to this prior assumption, here, we show that the tomato GLYCOALKALOID METABOLISM25 (GAME25), a short-chain dehydrogenase/reductase, catalyzes the first of three prospective reactions required to reduce the C-5,6 double bond in dehydrotomatidine to form tomatidine. The recombinant GAME25 enzyme displayed 3β-hydroxysteroid dehydrogenase/Δ5,4 isomerase activity not only on diverse steroidal alkaloid aglycone substrates but also on steroidal saponin aglycones. Notably, GAME25 down-regulation rerouted the entire tomato SGA repertoire toward the dehydro-SGAs branch rather than forming the typically abundant saturated α-tomatine derivatives. Overexpressing the tomato GAME25 in the tomato plant resulted in significant accumulation of α-tomatine in ripe fruit, while heterologous expression in cultivated eggplant generated saturated SGAs and atypical saturated steroidal saponin glycosides. This study demonstrates how a single scaffold modification of steroidal metabolites in plants results in extensive structural diversity and modulation of product toxicity.

Revisiting the Latency of Uridine Diphosphate-Glucuronosyltransferases (UGTs)-How Does the Endoplasmic Reticulum Membrane Influence Their Function?

Uridine diphosphate-glucuronosyltransferases (UGTs) are phase 2 conjugation enzymes mainly located in the endoplasmic reticulum (ER) of the liver and many other tissues, and can be recovered in artificial ER membrane preparations (microsomes). They catalyze glucuronidation reactions in various aglycone substrates, contributing significantly to the body’s chemical defense mechanism. There has been controversy over the last 50 years in the UGT field with respect to the explanation for the phenomenon of latency: full UGT activity revealed by chemical or physical disruption of the microsomal membrane. Because latency can lead to inaccurate measurements of UGT activity in vitro, and subsequent underprediction of drug clearance in vivo, it is important to understand the mechanisms behind this phenomenon. Three major hypotheses have been advanced to explain UGT latency: compartmentation, conformation, and adenine nucleotide inhibition. In this review, we discuss the evidence behind each hypothesis in depth, and suggest some additional studies that may reveal more information on this intriguing phenomenon.

Structural characterization of O- and C-glycosylating variants of the landomycin glycosyltransferase LanGT2.

The structures of the O-glycosyltransferase LanGT2 and the engineered, C-C bond-forming variant LanGT2S8Ac show how the replacement of a single loop can change the functionality of the enzyme. Crystal structures of the enzymes in complex with a nonhydrolyzable nucleotide-sugar analogue revealed that there is a conformational transition to create the binding sites for the aglycon substrate. This induced-fit transition was explored by molecular docking experiments with various aglycon substrates.

Crystal Structures of β-Primeverosidase in Complex with Disaccharide Amidine Inhibitors

β-Primeverosidase (PD) is a disaccharide-specific β-glycosidase in tea leaves. This enzyme is involved in aroma formation during the manufacturing process of oolong tea and black tea. PD hydrolyzes β-primeveroside (6-O-β-d-xylopyranosyl-β-d-glucopyranoside) at the β-glycosidic bond of primeverose to aglycone, and releases aromatic alcoholic volatiles of aglycones. PD only accepts primeverose as the glycone substrate, but broadly accepts various aglycones, including 2-phenylethanol, benzyl alcohol, linalool, and geraniol. We determined the crystal structure of PD complexes using highly specific disaccharide amidine inhibitors, N-β-primeverosylamidines, and revealed the architecture of the active site responsible for substrate specificity. We identified three subsites in the active site: subsite -2 specific for 6-O-β-d-xylopyranosyl, subsite -1 well conserved among β-glucosidases and specific for β-d-glucopyranosyl, and wide subsite +1 for hydrophobic aglycone. Glu-470, Ser-473, and Gln-477 act as the specific hydrogen bond donors for 6-O-β-d-xylopyranosyl in subsite -2. On the other hand, subsite +1 was a large hydrophobic cavity that accommodates various aromatic aglycones. Compared with aglycone-specific β-glucosidases of the glycoside hydrolase family 1, PD lacks the Trp crucial for aglycone recognition, and the resultant large cavity accepts aglycone and 6-O-β-d-xylopyranosyl together. PD recognizes the β-primeverosides in subsites -1 and -2 by hydrogen bonds, whereas the large subsite +1 loosely accommodates various aglycones. The glycone-specific activity of PD for broad aglycone substrates results in selective and multiple release of temporally stored alcoholic volatile aglycones of β-primeveroside.

Characterization of the zebrafish Ugt repertoire reveals a new class of drug-metabolizing UDP glucuronosyltransferases.

The zebrafish genome contains a gene superfamily of 40 Ugt genes that can be divided into Ugt1, Ugt2, and Ugt5 families. Because the encoded zebrafish UDP glucuronosyltransferase (UGT) proteins do not display orthologous relationships to any of the mammalian and avian UGT enzymes based on molecular phylogeny, it is difficult to predict their substrate specificity. Here, we mapped their tissue-specific expression patterns. We showed that the zebrafish UGT enzymes can be glycosylated. We determined their substrate specificity and catalytic activity toward diverse aglycone substrates. Specifically, we measured mRNA levels of each of the 40 zebrafish Ugt genes in 11 adult tissues and found that they are expressed in a tissue-specific manner. Moreover, functional analyses with the donor of UDP glucuronic acid (UDPGA) for each of the 40 zebrafish UGT proteins revealed their substrate specificity toward 10 important aglycones. In particular, UGT1A1, UGT1A7, and UGT1B1 displayed good glucuronidation activities toward most phenolic aglycones (4-methylumbelliferone, 4-nitrophenol, 1-naphthol, bisphenol A, and mycophenolic acid) and the two carboxylic acids (bilirubin and diclofenac). Importantly, some members of the UGT5, a novel UGT family identified recently, are capable of glucuronidating multiple aglycones with the donor cofactor of UDPGA. In particular, UGT5A5, UGT5B2, and UGT5E1 glucuronidate phenols and steroids with high specificity toward steroid hormones of estradiol and testosterone and estrogenic alkylphenols 4-tert-octylphenol. These results shed new insights into the mechanisms by which fish species defend themselves against vast numbers of xenobiotics via glucuronidation conjugations and may facilitate the establishment of zebrafish as a model vertebrate in toxicological, developmental, and pathologic studies.

Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG).

BackgroundLignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax.ResultsFive UGT genes belonging to the glycosyltransferases’ family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner.ConclusionWe have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis.

The specificity of α-glucosidase from Saccharomyces cerevisiae differs depending on the type of reaction: hydrolysis versus transglucosylation

Our investigation of the catalytic properties of Saccharomyces cerevisiae α-glucosidase (AGL) using hydroxybenzyl alcohol (HBA) isomers as transglucosylation substrates and their glucosides in hydrolytic reactions demonstrated interesting findings pertaining to the aglycon specificity of this important enzyme. AGL specificity increased from the para(p)- to the ortho(o)-HBA isomer in transglucosylation, whereas such AGL aglycon specificity was not seen in hydrolysis, thus indicating that the second step of the reaction (i.e., binding of the glucosyl acceptor) is rate-determining. To study the influence of substitution pattern on AGL kinetics, we compared AGL specificity, inferred from kinetic constants, for HBA isomers and other aglycon substrates. The demonstrated inhibitory effects of HBA isomers and their corresponding glucosides on AGL-catalyzed hydrolysis of p-nitrophenyl α-glucoside (PNPG) suggest that HBA glucosides act as competitive, whereas HBA isomers are noncompetitive, inhibitors. As such, we postulate that aromatic moieties cannot bind to an active site unless an enzyme-glucosyl complex has already formed, but they can interact with other regions of the enzyme molecule resulting in inhibition.

Component-based syntheses of trioxacarcin A, DC-45-A1 and structural analogues.

The trioxacarcins are polyoxygenated, structurally complex natural products that potently inhibit the growth of cultured human cancer cells. Here we describe syntheses of trioxacarcin A, DC-45-A1, and structural analogs by latestage, stereoselective glycosylation reactions of fully functionalized, differentially protected aglycon substrates. Key issues addressed in this work include the identification of an appropriate means to activate and protect each of the two 2-deoxysugar components, trioxacarcinose A and trioxacarcinose B, as well as a viable sequencing of the glycosidic couplings. The convergent, component-based sequence we present allows for rapid construction of structurally diverse, synthetic analogs that would be inaccessible by any other means, in amounts required to support biological evaluation. Analogs arising from modification of four of five modular components are assembled in 11 steps or fewer. The majority of these are found to be active in antiproliferative assays using cultured human cancer cells.

Enzymatic Glycosylation of Nonbenzoquinone Geldanamycin Analogs via Bacillus UDP-Glycosyltransferase

Geldanamycin (GM) is a naturally occurring anticancer agent isolated from several strains of Streptomyces hygroscopicus. However, its potential clinical utility is compromised by its severe toxicity and poor water solubility. For this reason, considerable efforts are under way to make new derivatives that have both good clinical efficacy and high water solubility. On the other hand, glycosylation is often a step that improves the water solubility and/or biological activity in many natural products of biosynthesis. Here, we report the facile production of glucose-conjugated nonbenzoquinone GM analogs using the Bacillus UDP-glycosyltransferase BL-C. Five aglycon substrates containing nonbenzoquinone aromatic rings were chosen to validate the in vitro glycosylation reaction. Putative glucoside compounds were determined through the presence of a product peak(s) and were also verified using LC/MS analyses. Further, the chemical structures of new glucoside compounds 6 and 7 were elucidated using spectroscopy data. These glucoside compounds showed a dramatic improvement in water solubility compared with that of the original aglycon, nonbenzoquinone GM.

UDP-Glucuronic Acid Binds First and the Aglycone Substrate Binds Second to Form a Ternary Complex in UGT1A9-Catalyzed Reactions, in Both the Presence and Absence of Bovine Serum Albumin

The presence of bovine serum albumin (BSA) largely modulates the enzyme kinetics parameters of the human UDP-glucuronosyltransferase (UGT) 1A9, increasing both the apparent aglycone substrate affinity of the enzyme and its limiting reaction velocity (Drug Metab Dispos 39:2117-2129, 2011). For a better understanding of the BSA effects and an examination of whether its presence changes the catalytic mechanism, we have studied the enzyme kinetics of 4-methylumbelliferone glucuronidation by UGT1A9 in the presence and absence of 0.1% BSA, using bisubstrate enzyme kinetic experiments, in both the forward and reverse directions, as well as product and dead-end inhibition. The combined results strongly suggest that the reaction mechanism of UGT1A9, and presumably other human UGTs as well, involves the formation of a compulsory-order ternary-complex, with UDP-α-d-glucuronic acid (UDPGA) as the first binding substrate. Based on the enzyme kinetic parameters measured for the forward and reverse reactions, the equilibrium constant of the overall reaction was calculated (Keq = 574) and the relative magnitudes of the reaction rate constants were elucidated. The inclusion of BSA in the bisubstrate kinetic experiments quantitatively changed the apparent enzyme kinetic parameters, presumably by removing internal inhibitors that bind to the binary enzyme-UDPGA (E-UDPGA) complex, as well as to the ternary E-UDPGA-aglycone complex. Nevertheless, the underlying compulsory-order ternary-complex mechanism with UDPGA binding first is the same in both the absence and presence of BSA. The results offer a novel understanding of UGT enzyme kinetic mechanism and BSA effects.

Sulfonation of glycopeptide antibiotics by sulfotransferase StaL depends on conformational flexibility of aglycone scaffold

Although glycopeptide antibiotics (GPAs), including vancomycin and teicoplanin, represent the most important class of anti-infective agents in the treatment of serious gram-positive bacterial infections, their usefulness is threatened by the emergence of resistant strains. GPAs are complex natural products consisting of a heptapeptide skeleton assembled via nonribosomal peptide synthesis and constrained through multiple crosslinks, with diversity resulting from enzymatic modifications by a variety of tailoring enzymes, which can be used to produce GPA analogues that could overcome antibiotic resistance. GPA-modifying sulfotransferases are promising tools for generating the unique derivatives. Despite significant sequence and structural similarities, these sulfotransferases modify distinct side chains on the GPA scaffold. To provide insight into the spatial diversity of modifications, we have determined the crystal structure of the ternary complex of bacterial sulfotransferase StaL with the cofactor product 3'-phosphoadenosine 5'-phosphate and desulfo-A47934 aglycone substrate. Desulfo-A47934 binds with the hydroxyl group on the 4-hydroxyphenylglycine in residue 1 directed toward the 3'-phosphoadenosine 5'-phosphate and hydrogen-bonded to the catalytic His67. Homodimeric StaL can accommodate GPA substrate in only one of the two active sites because of potential steric clashes. Importantly, the aglycone substrate demonstrates a flattened conformation, in contrast to the cup-shaped structures observed previously. Analysis of the conformations of this scaffold showed that despite the apparent rigidity due to crosslinking between the side chains, the aglycone scaffold displays substantial flexibility, important for enzymatic modifications by the GPA-tailoring enzymes. We also discuss the potential of using the current structural information in generating unique GPA derivatives.

Bovine Serum Albumin Decreases Km Values of Human UDP-Glucuronosyltransferases 1A9 and 2B7 and Increases Vmax Values of UGT1A9

The human UDP-glucuronosyltransferase (UGT) enzymes UGT1A9 and UGT2B7 play important roles in the hepatic glucuronidation of many drugs. The presence of bovine serum albumin (BSA) during in vitro assays was recently reported to lower the K(m) values of both these UGTs for their aglycone substrates without affecting the corresponding V(max) values. Nonetheless, using the specific substrates entacapone and zidovudine (AZT) for UGT1A9 and UGT2B7, respectively, and using an improved ultrafiltration method for measuring drug binding to BSA and to biological membranes, we found that the presence of BSA during the glucuronidation reaction leads to a large increase in the V(max) value of UGT1A9, in addition to lowering its K(m) value. On the other hand, in the case of UGT2B7, our results agree with the previously described effect of BSA, namely lowering the K(m) value without a large effect on the enzyme's V(max) value. The unexpected BSA effect on UGT1A9 was independent of the expression system because it was found in a recombinant enzyme that was expressed in baculovirus-infected insect cells as well as in the native enzyme in human liver microsomes. Moreover, the effect of BSA on the kinetics of 4-methylumbelliferone glucuronidation by recombinant UGT1A9 was similar to its effect on entacapone glucuronidation. Contrary to the aglycone substrates, the effect of BSA on the apparent K(m) of UGT1A9 for the cosubstrate UDP-α-D-glucuronic acid was nonsignificant. Our findings call for further investigations of the BSA effects on different UGTs and the inhibitors that it may remove.

PARTIAL PURIFICATION AND CHARACTERIZATION OF β-GLUCOSIDASE FROM TEA SHOOT

β-glucosidase was purified from the shoots of Camellia sinensis cv. Asha from Kangra valley following acetone precipitation, hydroxyapatite, gel filtration and ultrafiltration to apparent homogeneity. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of purified enzyme indicated a dimer with a larger subunit of ∼63 kD and a smaller subunit of ∼36 kD, molecular mass of ∼99 kD. The native form was confirmed by nondenaturating polyacrylamide gel electrophoresis. Optimum pH was 6.2, optimum temperature within 45–50C. β-glucosidase exhibited Km value of 0.16 mM and kcat of 11,000/min with the substrate p-nitrophenyl-β-D-glucopyranoside (pNpGlc) at 37C, pH 6.2. Substrate staining with 6-bromo-2-napthyl-β-D-glucopyranoside ruled out the possibility of any isozymic forms. The purified enzyme showed specificity toward pNpGlc and equivalent activity toward p-nitrophenyl-β-D-fucopyranoside. The enzyme liberated geraniol, linalool, phenylethanol, benzyl alcohol on incubation with tea shoot aqueous glycosidic extract. Highest activity was observed toward pNpGlc (100%), followed by p-nitrophenyl-β-D-mannopyranoside (79%) and p-nitrophenyl-β-D-fucopyranoside (75%), p-nitrophenyl-β-D-galactopyranoside (64%) etc., implying highly active β-glucosidase among other glycosidases. PRACTICAL APPLICATIONS β-glucosidases are reported to have transglycosylation activity. Conjugation of flavor volatiles as water-soluble glycosides provides an alternate means of biochemical trapping and storage of the volatile flavor compounds. The purified enzyme can be used for exogenous addition of glycosides and enzyme preparation to stimulate liberation of flavor volatiles for improving flavor quality of foods including tea, beverages and wine. Structural and functional analysis of protein gives a better understanding of catalytic mechanism and aglycone substrate specificity of β-glucosidase.

Reconstitution of antibiotics glycosylation by domain exchanged chimeric glycosyltransferase

Glycosyltransferase (GT), which catalyzes the transfer of nucleotide-activated sugar to aglycone, is the key enzyme for the preparation of a variety of glycosylated natural products. In this study, we report that the substrate specificity of GTs can be altered via domain exchanging accompanied by rationally designed peptide linkers. Comprehensive similarity searches were first conducted based upon the amino acid sequences of antibiotic GTs. The phylogenetic tree displays a distinct separation of 25 antibiotic GTs into three groups according to their aglycone substrates. Two different groups of GTs, KanF (aminoglycoside) and GtfE (glycopeptide) were employed for domain exchanging of GT-B fold enzyme. In order to determine linker peptide sequences, the secondary structures of KanF and GtfE were carefully evaluated via 3D-PSSM. The N-terminal domain of KanF and the C-terminal domain of GtfE were fused with rationally designed linker sequences. The hybrid GT, referred to as KE chimera, showed hybrid activity toward dTDP- d-glucose and 2-deoxystreptamine. Moreover, the key catalytic amino acid residues of KE chimera were investigated via protein modeling and docking simulations.

Expanding substrate specificity of GT‐B fold glycosyltransferase via domain swapping and high‐throughput screening

Glycosyltransferases (GTs) are crucial enzymes in the biosynthesis and diversification of therapeutically important natural products, and the majority of them belong to the GT-B superfamily, which is composed of separate N- and C-domains that are responsible for the recognition of the sugar acceptor and donor, respectively. In an effort to expand the substrate specificity of GT, a chimeric library with different crossover points was constructed between the N-terminal fragments of kanamycin GT (kanF) and the C-terminal fragments of vancomycin GT (gtfE) genes by incremental truncation method. A plate-based pH color assay was newly developed for the selection of functional domain-swapped GTs, and a mutant (HMT31) with a crossover point (N-kanF-669 bp and 753 bp-gtfE-C) for domain swapping was screened. The most active mutant HMT31 (50 kDa) efficiently catalyzed 2-DOS (aglycone substrate for KanF) glucosylation using dTDP-glucose (glycone substrate for GtfE) with k(cat)/K(m) of 162.8 +/- 0.1 mM(-1) min(-1). Moreover, HMT31 showed improved substrate specificity toward seven more NDP-sugars. This study presents a domain swapping method as a potential means to glycorandomization toward various syntheses of 2-DOS-based aminoglycoside derivatives.