Wanyi Guan

Discovery of Glycosyltransferases Using Carbohydrate Arrays and Mass Spectrometry

Glycosyltransferases (GTs) catalyze the reaction between an activated sugar donor and an acceptor to form a new glycosidic linkage. GTs are responsible for the assembly of oligosaccharides in vivo and are also important for the in vitro synthesis of these biomolecules. However, the functional identification and characterization of new GTs are both difficult and tedious. This paper describes an approach that combines arrays of reactions on an immobilized array of acceptors with analysis by mass spectrometry to screen putative GTs. A total of 14,280 combinations of GT, acceptor and donor in four buffer conditions were screened and led to the identification and characterization of four new GTs. This work is significant because it provides a label-free method for the rapid functional annotation of putative enzymes.

Enzymatic route to preparative-scale synthesis of UDP-GlcNAc/GalNAc, their analogues and GDP-fucose.

Enzymatic synthesis using glycosyltransferases is a powerful approach to building polysaccharides with high efficiency and selectivity. Sugar nucleotides are fundamental donor molecules in enzymatic glycosylation reactions by Leloir-type glycosyltransferases. The applications of these donors are restricted by their limited availability. In this protocol, N-acetylglucosamine (GlcNAc)/N-acetylgalactosamine (GalNAc) are phosphorylated by N-acetylhexosamine 1-kinase (NahK) and subsequently pyrophosphorylated by N-acetylglucosamine uridyltransferase (GlmU) to give UDP–GlcNAc/GalNAc. Other UDP–GlcNAc/GalNAc analogues can also be prepared depending on the tolerance of these enzymes to the modified sugar substrates. Starting from l-fucose, GDP–fucose is constructed by one bifunctional enzyme l-fucose pyrophosphorylase (FKP) via two reactions.

Substrate specificity of N-acetylhexosamine kinase towards N-acetylgalactosamine derivatives

We report herein a bacterial N-acetylhexosamine kinase, NahK, with broad substrate specificity towards structurally modified GalNAc analogues, and the production of a GalNAc-1-phosphate library using this kinase.

Characterization and synthetic application of a novel β1,3-galactosyltransferase from Escherichia coli O55:H7

A beta1,3-galactosyltransferase (WbgO) was identified in Escherichia coli O55:H7. Its function was confirmed by radioactive activity assay and structure analysis of the disaccharide synthesized with the recombinant enzyme. WbgO requires a divalent metal ion, either Mn(2+) or Mg(2+), for its activity and is active between pH 6.0-8.0 with a pH optimum of 7.0. N-acetylglucosamine (GlcNAc) and oligosaccharides with GlcNAc at the non-reducing end were shown to be its preferred substrates and it can be used for the synthesis of type 1 glycan chains from these substrates. Together with a recombinant bacterial GlcNAc-transferase, benzyl beta-lacto-N-tetraoside was synthesized with the purified WbgO to demonstrate the synthetic utility of WbgO.

Wide sugar substrate specificity of galactokinase from Streptococcus pneumoniae TIGR4.

Galactokinases (GALK) have attracted significant research attention for their potential application in the enzymatic synthesis of unique sugar phosphates. The galactokinase (GalKSpe4) cloned from Streptococcus pneumoniae TIGR4 had a temperature optimum of 45°C, and a pH optimum of 8.0. The substrate specificity and kinetics studies revealed that GalKSpe4 had moderate activity toward glucose, in contrast with very low or no activity observed in other previously reported GALKs. Most interestingly, GalKSpe4 exhibited activity for GalNAc, which had never been recorded in other GALKs found by now. This is the first time to report that bacterial GALK can recognize GalNAc.

Highly efficient synthesis of UDP-GalNAc/GlcNAc analogues with promiscuous recombinant human UDP-GalNAc pyrophosphorylase AGX1.

2-N-Acetamidosugars,[1] N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc), are prevalent in living organisms. They are key building blocks of glycosaminoglycans,[2] glycoproteins,[3] and glycolipids,[4] and are indispensable in glycoconjugate-involved cell communication and signaling.[4,5] In this context, we presumed that the analogues of the above-mentioned biologically essential amino sugars are valuable tools, either to unveil the metabolic pathways endowed by bioorthogonal groups,[6] or to expand the diversity of glycoconjugates with uncommon or non-natural sugars. Due to the structural complexity of glycans, chemical synthesis of glycoconjugates is an arduous task with tedious protection/deprotection, low yield and generally poor selectivity.[7] Therefore, enzymatic approach, most commonly Leloir-type glycosyltransferases, which transfer monosaccharide from the corresponding sugar nucleotide donor to an acceptor with high efficiency and selectivity, is an attractive alternative.[8] This means naturally occurring sugar nucleotides, as well as structural analogues are of paramount importance as substrates for enzymatic synthesis of oligosaccharides and glycoconjugates. Chemical synthesis of sugar nucleotides generally exploits the formation of the pyro-phosphate linkage from smaller building blocks, however, it was normally plagued with long reaction time, low overall yield and very strict reaction conditions.[9] On the other hand, enzymatic approach following the biosynthetic pathways requires multiple enzyme systems and protein engineering which also restricts the production of sugar nucleotides.[10] The natural donor molecules for GalNAc/GlcNAc are uridine 5′-diphospho-GalNAc/GlcNAc (UDP-GalNAc/ GlcNAc), and previously, we have synthesized eight UDP-GlcNAc analogues and three UDP-GalNAc analogues using recombinant Escherichia coli N-acetylglucosamine 1-phosphate uridylyltransferase (GlmU)[11] with moderate yields (10–65%) in a relatively large scale. Unfortunately, some sugar 1-phosphate (sugar-1-P) analogues, mostly GalNAc-1-P analogues, were not accepted by this enzyme. In order to overcome the narrow substrate specificity of GlmU towards GalNAc-1-P analogues and also enrich our sugar nucleotide analogue library, we explored the potential of human UDP-GalNAc pyrophosphorylase (AGX1), which had been applied to synthesize UDP-N-azidoacetylgalactosamine (UDP-GalNAz) in good yield but very small scale.[12] GalNAc-1-P/GlcNAc-1-P and their analogues used as substrates for sugar nucleotide preparation were produced chemoenzymatically as previously described.[13] The sugar nucleotides were obtained through uridylyl transfer reactions catalyzed by recombinant AGX1, and yeast inorganic pyro-phosphatase which was first employed in sugar nucleotide synthesis by Elling and Bulter[14] was also used here to drive the coupling reaction forward by degrading the byproduct PPi (Scheme 1). Reactions were monitored by thin-layer chromatography (TLC) and were terminated when complete consumption of sugar-1-Ps was observed. All sugar nucleotides were purified sequentially by anion exchange chromatography and, for desalting purpose, size exclusion chromatography (see Supporting Information). Scheme 1 General synthetic scheme of UDP-GalNAc/GlcNAc analogues; NahK=N-acetylhexosamine 1-kinase. Nine GalNAc-1-P analogues and five GlcNAc-1-P analogues (Table 1),[15,16] many of which had low yield or no reaction with GlmU,[11] were tested in this study. As shown in Table 1, AGX1 exhibited good tolerance towards both GalNAc- and GlcNAc-based structures. Eleven out of fourteen compounds were converted by AGX1 into the corresponding sugar nucleotides in preparative scale. The natural substrates GalNAc-1-P, GlcNAc-1-P (entries 1 and 10), and the 4-deoxy version (entry 8) were recognized at similar level, excluding the role of 4-hydroxyl group in substrate binding. However, the axial-4-azido entry 9 was non-isolable and only detected by TLC and MS, probably resulting from the larger substituent than hydroxyl. Table 1 Synthesis of UDP-GalNAc/GlcNAc utilizing AGX1 and GlmU.[11] Unlike GlmU, which differentiated GalNAc-1-P and GlcNAc-1-P analogues with bigger N-acyl modifications, AGX1 was only slightly affected by the bulkiness of N-acyl groups in both GalNAc-1-P and GlcNAc-1-P analogues. Briefly, GalNPr/GlcNPr-1-P (entry 2 and 11) and GalNAz/GlcNAz-1-P (entry 4 and 12) with relatively smaller N-propionyl and N-azidoacetyl groups were accepted with good conversion yield (>50%). GalNBu-1-P (entry 3) with a bulkier N-butyryl group had lower yield (44%) and longer reaction time, while a bulky N-benzoyl group prevents both GalNBz-1-P/GlcNBz-1-P to be accepted (entry 5 and 13). AGX1 also shows good tolerance to 6-modified GalNAc/ GlcNAc-1-P analogues. For example, not only 6-deoxy-GalNAc-1-P (entry 6) but also 6-azido-GalNAc-1-P and 6-azido-GlcNAc-1-P (entry 7 and 14) were taken by AGX1 to construct the corresponding UDP-sugars with good yields (>60%). This is another exciting result since GlmU only gave very poor yield for the 6-azido derivatives. The respective substrate specificity of AGX1 and GlmU is summarized in Table 2: First, both enzymes had limited acceptance for C-4 modifications. However, the 4-OH configuration did not affect the enzyme activity. Secondly, although very broad specificity of GlmU towards C-2 modified GlcNAc-1-P analogues was observed, including 2-azido-Glc-1-P and 2-keto-Glc-1-P,[17] it was not a good candidate for the preparation of C-2 and C-6 modified UDP-GalNAc analogues. Last but not least, we proved AGX1 as a better choice for synthesizing most UDP-GalNAc/GlcNAc analogues with C-2,4,6 modifications. Table 2 Summarized substrate specificity of AGX1 and GlmU. In summary, owing to the promiscuity of recombinant human UDP-GalNAc pyrophosphorylase (AGX1), we have successfully prepared C-2,4,6 modified UDP-GalNAc/ GlcNAc analogues on a preparative scale with good yields. We thus avoided having to design and obtain GlmU mutants to accommodate the unacceptable substrate structures. We believe our library of UDP-GalNAc/GlcNAc analogues could greatly facilitate our investigation into the substrate specificity of various glycosyltransferases as well as provide a significant step towards natural product glycodiversification.

Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP)

Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP.

Combining carbochips and mass spectrometry to study the donor specificity for the Neisseria meningitidis β1,3-N-acetylglucosaminyltransferase LgtA.

A library of 11 UDP-N-acetylglucosamine analogs were rapidly screened for their activities as donors for the Neisseria meningitidis β1,3-N-acetylglucosaminyltransferase (LgtA) by direct on-chip reaction and detection with SAMDI-TOF mass spectrometry. Six of the analogs were active in this assay and were analyzed by SAMDI to characterize the kinetics toward LgtA. The analysis revealed that substitutions on C-2, C-4, and C-6 affect the activity of the donors, with bulky groups at these positions decreasing affinity of the donors for the enzyme, and also revealed that activity is strongly affected by the stereochemistry at C-3, but not C-4, of the donor. The study is also significant because it demonstrates that SAMDI can be used to both profile glycosyltransferase activities and to provide a quantitative assessment of enzyme activity.

Altered architecture of substrate binding region defines the unique specificity of UDP‐GalNAc 4‐epimerases

UDP‐hexose 4‐epimerases play a pivotal role in lipopolysaccharide (LPS) biosynthesis and Leloir pathway. These epimerases are classified into three groups based on whether they recognize nonacetylated UDP‐hexoses (Group 1), both N‐acetylated and nonacetylated UDP‐hexoses (Group 2) or only N‐acetylated UDP‐hexoses (Group 3). Although the catalysis has been investigated extensively, yet a definitive model rationalizing the substrate specificity of all the three groups on a common platform is largely lacking. In this work, we present the crystal structure of WbgU, a novel UDP‐hexose 4‐epimerase that belongs to the Group 3. WbgU is involved in biosynthetic pathway of the unusual glycan 2‐deoxy‐L‐altruronic acid that is found in the LPS of the pathogen Pleisomonas shigelloides. A model that defines its substrate specificity is proposed on the basis of the active site architecture. Representatives from all the three groups are then compared to rationalize their substrate specificity. This investigation reveals that the Group 3 active site architecture is markedly different from the “conserved scaffold” of the Group 1 and the Group 2 epimerases and highlights the interactions potentially responsible for the origin of specificity of the Group 3 epimerases toward N‐acetylated hexoses. This study provides a platform for further engineering of the UDP‐hexose 4‐epimerases, leads to a deeper understanding of the LPS biosynthesis and carbohydrate recognition by proteins. It may also have implications in development of novel antibiotics and more economic synthesis of UDP‐GalNAc and downstream products such as carbohydrate based vaccines.

Enzymatic synthesis and properties of uridine-5′-O-(2-thiodiphospho)-N-acetylglucosamine

This paper describes an enzymatic approach to obtain a thio-containing UDP-GlcNAc analog. We use an assay based on capture of the carbohydrate and analysis by mass spectrometry to quantitatively characterize the activity of this unnatural sugar donor in a LgtA-mediated glycosylation reaction.