A GH89 human α-N-acetylglucosaminidase (hNAGLU) homologue from gut microbe Bacteroides thetaiotaomicron capable of hydrolyzing heparosan oligosaccharides.

Carbohydrate-Active enZYme (CAZY) GH89 family enzymes catalyze the cleavage of terminal α-N-acetylglucosamine from glycans and glycoconjugates. Although structurally and mechanistically similar to the human lysosomal α-N-acetylglucosaminidase (hNAGLU) in GH89 which is involved in the degradation of heparan sulfate in the lysosome, the reported bacterial GH89 enzymes characterized so far have no or low activity toward α-N-acetylglucosamine-terminated heparosan oligosaccharides, the preferred substrates of hNAGLU. We cloned and expressed several soluble and active recombinant bacterial GH89 enzymes in Escherichia coli. Among these enzymes, a truncated recombinant α-N-acetylglucosaminidase from gut symbiotic bacterium Bacteroides thetaiotaomicron ∆22Bt3590 was found to catalyze the cleavage of the terminal α1-4-linked N-acetylglucosamine (GlcNAc) from a heparosan disaccharide with high efficiency. Heparosan oligosaccharides with lengths up to decasaccharide were also suitable substrates. This bacterial α-N-acetylglucosaminidase could be a useful catalyst for heparan sulfate analysis.

Microbial production of human milk oligosaccharide lactodifucotetraose.

Human milk oligosaccharides (HMOs) are potent bioactive compounds that modulate neonatal health and are of interest for development as potential drug treatments for adult diseases. The potential of these molecules, their limited access from natural sources, and difficulty in large-scale isolation of individual HMOs for studies and applications have motivated the development of chemical syntheses and in vitro enzymatic catalysis strategies. Whole cell biocatalysts are emerging as alternative self-regulating production platforms that have the potential to reduce the cost for enzymatic synthesis of HMOs. Whole cell biocatalysts for the production of short-chained, linear and small monofucosylated HMOs have been reported but those for fucosylated structures with higher complexity have not been explored. In this study, we established a strategy for producing a difucosylated HMO, lactodifucotetraose (LDFT), from lactose and L-fucose in Escherichia coli. We used two bacterial fucosyltransferases with narrow acceptor selectivity to drive the sequential fucosylation of lactose and intermediate 2'-fucosyllactose (2'-FL) to produce LDFT. Deletion of substrate degradation pathways that decoupled cellular growth from LDFT production, enhanced expression of native substrate transporters and modular induction of the genes in the LDFT biosynthetic pathway allowed complete conversion of lactose into LDFT and minor quantities of the side product 3-fucosyllactose (3-FL). Overall, 5.1 g/L of LDFT was produced from 3 g/L lactose and 3 g/L L-fucose in 24 h. Our results demonstrate promising applications of engineered microbial biosystems for the production of multi-fucosylated HMOs for biochemical studies.

L. pneumophila CMP-5,7-di-N-acetyllegionaminic acid synthetase (LpCLS)-involved chemoenzymatic synthesis of sialosides and analogues.

5,7-Di-N-acetyllegionaminic acid (Leg5,7Ac2) is a bacterial nonulosonic acid (NulO) analogue of sialic acids, an important class of monosaccharides in mammals and in some bacteria. To develop efficient one-pot multienzyme (OPME) glycosylation systems for synthesizing Leg5,7Ac2-glycosides, Legionella pneumophila cytidine 5'-monophosphate (CMP)-Leg5,7Ac2 synthetase (LpCLS) was cloned and characterized. It was successfully used in producing Leg5,7Ac2-glycosides from chemoenzymatically synthesized Leg5,7Ac2 using a one-pot two-enzyme system or from its chemically synthesized six-carbon monosaccharide precursor 2,4-diacetamido-2,4,6-trideoxymannose (6deoxyMan2,4diNAc) in a one-pot three-enzyme system. In addition, LpCLS was shown to tolerate Neu5Ac7NAc, a C9-hydroxyl analogue of Leg5,7Ac2 and also a stable analogue of 7-O-acetylneuraminic acid (Neu5,7Ac2), to allow OPME synthesis of the corresponding α2-3-linked sialosides, from chemically synthesized six-carbon monosaccharide precursor 4-N-acetyl-4-deoxy-N-acetylmannosamine (ManNAc7NAc).

Catalytic Cycle of Neisseria meningitidis CMP-Sialic Acid Synthetase Illustrated by High-Resolution Protein Crystallography.

Cytidine 5'-monophosphate (CMP)-sialic acid synthetase (CSS) is an essential enzyme involved in the biosynthesis of carbohydrates and glycoconjugates containing sialic acids, a class of α-keto acids that are generally terminal key recognition residues by many proteins that play important biological and pathological roles. The CSS from Neisseria meningitidis (NmCSS) has been commonly used with other enzymes such as sialic acid aldolase and/or sialyltransferase in synthesizing a diverse array of compounds containing sialic acid or its naturally occurring and non-natural derivatives. To better understand its catalytic mechanism and substrate promiscuity, four NmCSS crystal structures trapped at various stages of the catalytic cycle with bound substrates, substrate analogues, and products have been obtained and are presented here. These structures suggest a mechanism for an "open" and "closed" conformational transition that occurs as sialic acid binds to the NmCSS/cytidine-5'-triphosphate (CTP) complex. The closed conformation positions critical residues to help facilitate the nucleophilic attack of sialic acid C2-OH to the α-phosphate of CTP, which is also aided by two observed divalent cations. Product formation drives the active site opening, promoting the release of products.

Engineer P. multocida Heparosan Synthase 2 (PmHS2) for Size-Controlled Synthesis of Longer Heparosan Oligosaccharides.

Pasteurella multocida heparosan synthase 2 (PmHS2) is a dual-function polysaccharide synthase having both α1-4-N-acetylglucosaminyltransferase (α1-4-GlcNAcT) and ß1-4-glucuronyltransferase (ß1-4-GlcAT) activities located in two separate catalytic domains. We found that removing PmHS2 N-terminal 80-amino acid residues improved enzyme stability and expression level while retaining its substrate promiscuity. We also identified the reverse glycosylation activities of PmHS2 which complicated its application in size-controlled synthesis of oligosaccharides longer than hexasaccharide. Engineered Δ80PmHS2 single-function-glycosyltransferase mutants Δ80PmHS2_D291N (α1-4-GlcNAcT lacking both forward and reverse ß1-4-GlcAT activities) and Δ80PmHS2_D569N (ß1-4-GlcAT lacking both forward and reverse α1-4-GlcNAcT activities) were designed and showed to minimize side product formation. They were successfully used in a sequential one-pot multienzyme (OPME) platform for size-controlled high-yield production of oligosaccharides up to decasaccharide. The study draws attention to the consideration of reverse glycosylation activities of glycosyltransferases, including polysaccharide synthases, when applying them in the synthesis of oligosaccharides and polysaccharides. The mutagenesis strategy has the potential to be extended to other multifunctional polysaccharide synthases with reverse glycosylation activities to generate catalysts with improved synthetic efficiency.

Strategies for chemoenzymatic synthesis of carbohydrates.

Carbohydrates are structurally complex but functionally important biomolecules. Therefore, they have been challenging but attractive synthetic targets. While substantial progress has been made on advancing chemical glycosylation methods, incorporating enzymes into carbohydrate synthetic schemes has become increasingly practical as more carbohydrate biosynthetic and metabolic enzymes as well as their mutants with synthetic application are identified and expressed for preparative and large-scale synthesis. Chemoenzymatic strategies that integrate the flexibility of chemical derivatization with enzyme-catalyzed reactions have been extremely powerful. Briefly summarized here are our experiences on developing one-pot multienzyme (OPME) systems and representative chemoenzymatic strategies from others using glycosyltransferase-catalyzed reactions for synthesizing diverse structures of oligosaccharides, polysaccharides, and glycoconjugates. These strategies allow the synthesis of complex carbohydrates including those containing naturally occurring carbohydrate postglycosylational modifications (PGMs) and non-natural functional groups. By combining these srategies with facile purification schemes, synthetic access to the diverse space of carbohydrate structures can be automated and will not be limited to specialists.

A Bacterial ß1-3-Galactosyltransferase Enables Multigram-Scale Synthesis of Human Milk Lacto-N-tetraose (LNT) and Its Fucosides.

ß1-3-Linked galactosides such as Galß1‒3GlcNAcßOR are common carbohydrate motifs found in human milk oligosaccharides (HMOSs), glycolipids, and glycoproteins. Efficient and scalable enzymatic syntheses of these structures have proven challenging due to the lack of access to a highly active ß1‒3-galactosyltransferase (ß3GalT) in large amounts. Previously reported E. coli ß3GalT (EcWbgO) has been identified as a limiting factor for producing a ß1-3-galactose-terminated human milk oligosaccharide lacto-N-tetraose (LNT) by fermentation. Here we report the identification of an EcWbgO homolog from C. violaceum (Cvß3GalT) which showed a high efficiency in catalyzing the formation of LNT from lacto-N-triose (LNT II). With the highly active Cvß3GalT, multigram-scale (>10 gram) synthesis of LNT from lactose was achieved using a sequential one-pot multienzyme (OPME) glycosylation process. The access to Cvß3GalT enabled enzymatic synthesis of several fucosylated HMOSs with or without further sialylation including LNFP II, S-LNF II, LNDFH I, LNFP V, and DiFuc-LNT. Among these, LNFP V and DiFuc-LNT would not be accessible by enzymatic synthesis if an active ß3GalT were not available.

Triazole-linked transition state analogs as selective inhibitors against V. cholerae sialidase.

Sialidases or neuraminidases are enzymes that catalyze the cleavage of terminal sialic acids from oligosaccharides and glycoconjugates. They play important roles in bacterial and viral infection and have been attractive targets for drug development. Structure-based drug design has led to potent inhibitors against neuraminidases of influenza A viruses that have been used successfully as approved therapeutics. However, selective and effective inhibitors against bacterial and human sialidases are still being actively pursued. Guided by crystal structural analysis, several derivatives of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en or DANA) were designed and synthesized as triazole-linked transition state analogs. Inhibition studies revealed that glycopeptide analog E-(TriazoleNeu5Ac2en)-AKE and compound (TriazoleNeu5Ac2en)-A were selective inhibitors against Vibrio cholerae sialidase, while glycopeptide analog (TriazoleNeu5Ac2en)-AdE selectively inhibited Vibrio cholerae and A. ureafaciens sialidases.

Streamlined chemoenzymatic total synthesis of prioritized ganglioside cancer antigens.

A highly efficient streamlined chemoenzymatic strategy for total synthesis of four prioritized ganglioside cancer antigens GD2, GD3, fucosyl GM1, and GM3 from commercially available lactose and phytosphingosine is demonstrated. Lactosyl sphingosine (LacßSph) was chemically synthesized (on a 13 g scale), subjected to sequential one-pot multienzyme (OPME) glycosylation reactions with facile C18-cartridge purification, followed by improved acylation conditions to form target gangliosides, including fucosyl GM1 which has never been synthesized before.

α2-6-Neosialidase: A Sialyltransferase Mutant as a Sialyl Linkage-Specific Sialidase.

The lack of α2-6-linkage specific sialidases limits the structural and functional studies of sialic-acid-containing molecules. Photobacterium damselae α2-6-sialyltransferase (Pd2,6ST) was shown previously to have α2-6-specific, but weak, sialidase activity. Here, we develop a high-throughput blue-white colony screening method to identify Pd2,6ST mutants with improved α2-6-sialidase activity from mutant libraries generated by sequential saturation mutagenesis. A triple mutant (Pd2,6ST S232L/T356S/W361F) has been identified with 100-fold improved activity, high α2-6-sialyl linkage selectivity, and ability to cleave two common sialic acid forms, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). It is a valuable tool for sialoglycan structural analysis and functional characterization. The sequential saturation mutagenesis and screening strategy developed here can be explored to evolve other linkage-specific neoglycosidases from the corresponding glycosyltransferases.

A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di-N-acetyllegionaminic Acid-Containing Glycosides.

A chemoenzymatic synthon was designed to expand the scope of the chemoenzymatic synthesis of carbohydrates. The synthon was enzymatically converted into carbohydrate analogues, which were readily derivatized chemically to produce the desired targets. The strategy is demonstrated for the synthesis of glycosides containing 7,9-di-N-acetyllegionaminic acid (Leg5,7Ac2 ), a bacterial nonulosonic acid (NulO) analogue of sialic acid. A versatile library of α2-3/6-linked Leg5,7Ac2 -glycosides was built by using chemically synthesized 2,4-diazido-2,4,6-trideoxymannose as a chemoenzymatic synthon for highly efficient one-pot multienzyme (OPME) sialylation followed by downstream chemical conversion of the azido groups into acetamido groups. The syntheses required 10 steps from commercially available d-fucose and had an overall yield of 34-52 %, thus representing a significant improvement over previous methods. Free Leg5,7Ac2 monosaccharide was also synthesized by a sialic acid aldolase-catalyzed reaction.

Converting Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) to a regioselective α2-6-sialyltransferase by saturation mutagenesis and regioselective screening.

A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) for α2-6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective α2-6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activity compared to the wild-type PmST1. It retained the donor substrate promiscuity of the wild-type enzyme and was used in an efficient one-pot multienzyme (OPME) system to selectively catalyze the sialylation of the terminal galactose residue in a multigalactose-containing tetrasaccharide lacto-N-neotetraoside.

Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering.

Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate toward a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.

Glycosyltransferase engineering for carbohydrate synthesis.

Glycosyltransferases (GTs) are powerful tools for the synthesis of complex and biologically-important carbohydrates. Wild-type GTs may not have all the properties and functions that are desired for large-scale production of carbohydrates that exist in nature and those with non-natural modifications. With the increasing availability of crystal structures of GTs, especially those in the presence of donor and acceptor analogues, crystal structure-guided rational design has been quite successful in obtaining mutants with desired functionalities. With current limited understanding of the structure-activity relationship of GTs, directed evolution continues to be a useful approach for generating additional mutants with functionality that can be screened for in a high-throughput format. Mutating the amino acid residues constituting or close to the substrate-binding sites of GTs by structure-guided directed evolution (SGDE) further explores the biotechnological potential of GTs that can only be realized through enzyme engineering. This mini-review discusses the progress made towards GT engineering and the lessons learned for future engineering efforts and assay development.

Characterizing non-hydrolyzing Neisseria meningitidis serogroup A UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase using UDP-N-acetylmannosamine (UDP-ManNAc) and derivatives.

Neisseria meningitidis serogroup A non-hydrolyzing uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase (NmSacA) catalyzes the interconversion between UDP-GlcNAc and uridine 5'-diphosphate-N-acetylmannosamine (UDP-ManNAc). It is a key enzyme involved in the biosynthesis of the capsular polysaccharide [-6ManNAcα1-phosphate-]n of N. meningitidis serogroup A, one of the six serogroups (A, B, C, W-135, X, and Y) that account for most cases of N. meningitidis-caused bacterial septicemia and meningitis. N. meningitidis serogroup A is responsible for large epidemics in the developing world, especially in Africa. Here we report that UDP-ManNAc could be used as a substrate for C-terminal His6-tagged recombinant NmSacA (NmSacA-His6) in the absence of UDP-GlcNAc. NmSacA-His6 was activated by UDP-GlcNAc and inhibited by 2-acetamidoglucal and UDP. Substrate specificity study showed that NmSacA-His6 could tolerate several chemoenzymatically synthesized UDP-ManNAc derivatives as substrates although its activity was much lower than non-modified UDP-ManNAc. Homology modeling and molecular docking revealed likely structural determinants of NmSacA substrate specificity. This is the first detailed study of N. meningitidis serogroup A UDP-GlcNAc 2-epimerase.

Promiscuity of a modular polyketide synthase towards natural and non-natural extender units.

Combinatorial biosynthesis approaches that involve modular type I polyketide synthases (PKSs) are proven strategies for the synthesis of polyketides. In general however, such strategies are usually limited in scope and utility due to the restricted substrate specificity of polyketide biosynthetic machinery. Herein, a panel of chemo-enzymatically synthesized acyl-CoA's was used to probe the promiscuity of a polyketide synthase. Promiscuity determinants were dissected, revealing that the KS is remarkably tolerant to a diverse array of extender units, while the AT likely discriminates between extender units that are native to the producing organism. Our data provides a clear blueprint for future enzyme engineering efforts, and sets the stage for harnessing extender unit promiscuity by employing various in vivo polyketide diversification strategies.