A high-throughput screening platform for enzymes active on mucin-type O-glycoproteins.

Mucin-type O-glycosylation is a post-translational modification present at the interface between cells where it has important roles in cellular communication. However, deciphering the function of O-glycoproteins and O-glycans can be challenging, especially as few enzymes are available for their assembly or selective degradation. Here, to address this deficiency, we developed a genetically encoded screening methodology for the discovery and engineering of the diverse classes of enzymes that act on O-glycoproteins. The method uses Escherichia coli that have been engineered to produce an O-glycosylated fluorescence resonance energy transfer probe that can be used to screen for O-glycopeptidase activity. Subsequent cleavage of the substrate by O-glycopeptidases provides a read-out of the glycosylation state of the probe, allowing the method to also be used to assay glycosidases and glycosyltransferases. We further show the potential of this methodology in the first ultrahigh-throughput-directed evolution of an O-glycopeptidase.

O-GlcNAc transferase modulates the cellular endocytosis machinery by controlling the formation of clathrin-coated pits.

Clathrin-mediated endocytosis (CME) controls the internalization and function of a wide range of cell surface proteins. CME occurs by the assembly of clathrin and many other proteins on the inner leaflet of the plasma membrane into clathrin-coated pits (CCPs). These structures recruit specific cargo destined for internalization, generate membrane curvature, and in many cases undergo scission from the plasma membrane to yield intracellular vesicles. The diversity of functions of cell surface proteins controlled via internalization by CME may suggest that regulation of CCP formation could be effective to allow cellular adaptation under different contexts. Of interest is how cues derived from cellular metabolism may regulate CME, given the reciprocal role of CME in controlling cellular metabolism. The modification of proteins with O-linked ß-GlcNAc (O-GlcNAc) is sensitive to nutrient availability and may allow cellular adaptation to different metabolic conditions. Here, we examined how the modification of proteins with O-GlcNAc may control CCP formation and thus CME. We used perturbation of key enzymes responsible for protein O-GlcNAc modification, as well as specific mutants of the endocytic regulator AAK1 predicted to be impaired for O-GlcNAc modification. We identify that CCP initiation and the assembly of clathrin and other proteins within CCPs are controlled by O-GlcNAc protein modification. This reveals a new dimension of regulation of CME and highlights the important reciprocal regulation of cellular metabolism and endocytosis.

Chitin-Active Lytic Polysaccharide Monooxygenases Are Rare in Cellulomonas Species.

Cellulomonas flavigena is a saprotrophic bacterium that encodes, within its genome, four predicted lytic polysaccharide monooxygenases (LPMOs) from Auxiliary Activity family 10 (AA10). We showed previously that three of these cleave the plant polysaccharide cellulose by oxidation at carbon-1 (J. Li, L. Solhi, E.D. Goddard-Borger, Y. Mattieu et al., Biotechnol Biofuels 14:29, 2021, https://doi.org/10.1186/s13068-020-01860-3). Here, we present the biochemical characterization of the fourth C. flavigena AA10 member (CflaLPMO10D) as a chitin-active LPMO. Both the full-length CflaLPMO10D-Carbohydrate-Binding Module family 2 (CBM2) and catalytic module-only proteins were produced in Escherichia coli using the native general secretory (Sec) signal peptide. To quantify chitinolytic activity, we developed a high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) method as an alternative to the established hydrophilic interaction liquid ion chromatography coupled with UV detection (HILIC-UV) method for separation and detection of released oxidized chito-oligosaccharides. Using this method, we demonstrated that CflaLPMO10D is strictly active on the ß-allomorph of chitin, with optimal activity at pH 5 to 6 and a preference for ascorbic acid as the reducing agent. We also demonstrated the importance of the CBM2 member for both mediating enzyme localization to substrates and prolonging LPMO activity. Together with previous work, the present study defines the distinct substrate specificities of the suite of C. flavigena AA10 members. Notably, a cross-genome survey of AA10 members indicated that chitinolytic LPMOs are, in fact, rare among Cellulomonas bacteria. IMPORTANCE Species from the genus Cellulomonas have a long history of study due to their roles in biomass recycling in nature and corresponding potential as sources of enzymes for biotechnological applications. Although Cellulomonas species are more commonly associated with the cleavage and utilization of plant cell wall polysaccharides, here, we show that C. flavigena produces a unique lytic polysaccharide monooxygenase with activity on ß-chitin, which is found, for example, in arthropods. The limited distribution of orthologous chitinolytic LPMOs suggests adaptation of individual cellulomonads to specific nutrient niches present in soil ecosystems. This research provides new insight into the biochemical specificity of LPMOs in Cellulomonas species and related bacteria, and it raises new questions about the physiological function of these enzymes.

Mammalian sialyltransferases allow efficient Escherichia coli-based production of mucin-type O-glycoproteins but can also transfer Kdo.

The prospect of producing human-like glycoproteins in bacteria is becoming attractive as an alternative to already-established but costly mammalian cell expression systems. We previously described an Escherichia coli expression platform that uses a dual-plasmid approach to produce simple mucin type O-glycoproteins: one plasmid encoding the target protein and another O-glycosylation machinery. Here, we expand the capabilities of our platform to carry out sialylation and demonstrate the high-yielding production of human interferon α2b and human growth hormone bearing mono- and disialylated T-antigen glycans. This is achieved through engineering an E. coli strain to produce CMP-Neu5Ac and introducing various α-2,3- and α-2,6 mammalian or bacterial sialyltransferases into our O-glycosylation operons. We further demonstrate that mammalian sialyltransferases, including porcine ST3Gal1, human ST6GalNAc2 and human ST6GalNAc4, are very effective in vivo and outperform some of the bacterial sialyltransferases tested, including Campylobacter jejuni Cst-I and Cst-II. In the process, we came upon a way of modifying T-Antigen with Kdo, using a previously uncharacterised Kdo-transferase activity of porcine ST3Gal1. Ultimately, the heterologous expression of mammalian sialyltransferases in E. coli shows promise for the further development of bacterial systems in therapeutic glycoprotein production.

Quantifying Carbohydrate-Active Enzyme Activity with Glycoprotein Substrates Using Electrospray Ionization Mass Spectrometry and Center-of-Mass Monitoring.

Carbohydrate-active enzymes (CAZymes) play critical roles in diverse physiological and pathophysiological processes and are important for a wide range of biotechnology applications. Kinetic measurements offer insight into the activity and substrate specificity of CAZymes, information that is of fundamental interest and supports diverse applications. However, robust and versatile kinetic assays for monitoring the kinetics of intact glycoprotein and glycolipid substrates are lacking. Here, we introduce a simple but quantitative electrospray ionization mass spectrometry (ESI-MS) method for measuring the kinetics of CAZyme reactions involving glycoprotein substrates. The assay, referred to as center-of-mass (CoM) monitoring (CoMMon), relies on continuous (real-time) monitoring of the CoM of an ensemble of glycoprotein substrates and their corresponding CAZyme products. Notably, there is no requirement for calibration curves, internal standards, labeling, or mass spectrum deconvolution. To demonstrate the reliability of CoMMon, we applied the method to the neuraminidase-catalyzed cleavage of N-acetylneuraminic acid (Neu5Ac) residues from a series of glycoproteins of varying molecular weights and degrees of glycosylation. Reaction progress curves and initial rates determined with CoMMon are in good agreement (initial rates within ≤5%) with results obtained, simultaneously, using an isotopically labeled Neu5Ac internal standard, which enabled the time-dependent concentration of released Neu5Ac to be precisely measured. To illustrate the applicability of CoMMon to glycosyltransferase reactions, the assay was used to measure the kinetics of sialylation of a series of asialo-glycoproteins by a human sialyltransferase. Finally, we show how combining CoMMon and the competitive universal proxy receptor assay enables the relative reactivity of glycoprotein substrates to be quantitatively established.

Investigation of sequon engineering for improved O-glycosylation by the human polypeptide N-acetylgalactosaminyl transferase T2 isozyme and two orthologues.

We have been developing bacterial expression systems for human mucin-type O-glycosylation on therapeutic proteins, which is initiated by the addition of α-linked GalNAc to serine or threonine residues by enzymes in the GT-27 family of glycosyltransferases. Substrate preference across different isoforms of this enzyme is influenced by isoform-specific amino acid sequences at the site of glycosylation, which we have exploited to engineer production of Core 1 glycan structures in bacteria on human therapeutic proteins. Using RP-HPLC with a novel phenyl bonded phase to resolve intact protein glycoforms, the effect of sequon mutation on O-glycosylation initiation was examined through in vitro modification of the naturally O-glycosylated human interferon α-2b, and a sequon engineered human growth hormone. As part of the development of our glycan engineering in the bacterial expression system we are surveying various orthologues of critical enzymes to ensure complete glycosylation. Here we present an in vitro enzyme kinetic profile of three related GT-27 orthologues on natural and engineered sequons in recombinant human interferon α2b and human growth hormone where we show a significant change in kinetic properties with the amino acid changes. It was found that optimizing the protein substrate amino acid sequence using Isoform Specific O-Glycosylation Prediction (ISOGlyP, http://isoglyp.utep.edu/index.php) resulted in a measurable increase in kcat/KM, thus improving glycosylation efficiency. We showed that the Drosophila orthologue showed superior activity with our human growth hormone designed sequons compared with the human enzyme.

7-Fluorosialyl Glycosides Are Hydrolysis Resistant but Readily Assembled by Sialyltransferases Providing Easy Access to More Metabolically Stable Glycoproteins.

The maintenance of therapeutic glycoproteins within the circulatory system is associated, in large part, with the integrity of sialic acids as terminal sugars on the glycans. Glycoprotein desialylation, either by spontaneous cleavage or through host sialidases, leads to protein clearance, mainly through the liver. Thus, the installation of minimally modified sialic acids that are hydrolysis-resistant yet biologically equivalent should lead to increased circulatory half-lives and improved pharmacokinetic profiles. Here we describe the chemoenzymatic synthesis of CMP-sialic acid sugar donors bearing fluorine atoms at the 7-position, starting from the corresponding 4-deoxy-4-fluoro-N-acetylhexosamine precursors. For the derivative with natural stereochemistry we observe efficient glycosyl transfer by sialyltransferases, along with improved stability of the resultant 7-fluorosialosides toward spontaneous hydrolysis (3- to 5-fold) and toward cleavage by GH33 sialidases (40- to 250-fold). Taking advantage of the rapid transfer of 7-fluorosialic acid by sialyltransferases, we engineered the O-glycan of Interferon α-2b and the N-glycans of the therapeutic glycoprotein α1-antitrypsin. Studies of the uptake of the glyco-engineered α1-antitrypsin by HepG2 liver cells demonstrated the bioequivalence of 7-fluorosialic acid to sialic acid in suppressing interaction with liver cell lectins. In vivo pharmacokinetic studies reveal enhanced half-life of the protein decorated with 7-fluorosialic acid relative to unmodified sialic acid in the murine circulatory system. 7-Fluorosialylation therefore offers considerable promise as a means of prolonging circulatory half-lives of glycoproteins and may pave the way toward biobetters for therapeutic use.

Development of BODIPY labelled sialic acids as sialyltransferase substrates for direct detection of terminal galactose on N- and O-linked glycans.

Glycans on proteins and cell surfaces are useful biomarkers for determining functional interactions with glycan binding proteins, potential disease states, or indeed level of differentiation. The ability to rapidly and sensitively detect or tag specific glycans on proteins provides a diagnostic tool with wide application in chemical glycobiology. The monosaccharide N-acetylneuraminic acid (sialic acid) is a key player in these interactions and the manipulation and control of sialylation levels has been an important research focus, particularly in the development of therapeutic proteins. Using sialyltransferases to tag specific glycans provides a rapid means of determining what types of glycans are present. We have synthesized two variants of sialic acid carrying the fluorophore BODIPY (4,4 -Difluoro-4-boro-3a,4a-diaza-s-indacene) and examined its use with several different sialyltransferases on a variety of protein substrates and cell surface glycans. Our data show that there are significant differences between various enzymes ability to transfer the labelled sialic acids, and that the type of N-glycan and target protein strongly influences this activity.

Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species.

BACKGROUND: The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). RESULTS: Here, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation. CONCLUSIONS: Together, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.

Energetic adaptations: Metabolic control of endocytic membrane traffic.

Endocytic membrane traffic controls the access of myriad cell surface proteins to the extracellular milieu, and thus gates nutrient uptake, ion homeostasis, signaling, adhesion and migration. Coordination of the regulation of endocytic membrane traffic with a cell's metabolic needs represents an important facet of maintenance of homeostasis under variable conditions of nutrient availability and metabolic demand. Many studies have revealed intimate regulation of endocytic membrane traffic by metabolic cues, from the specific control of certain receptors or transporters, to broader adaptation or remodeling of the endocytic membrane network. We examine how metabolic sensors such as AMP-activated protein kinase, mechanistic target of rapamycin complex 1 and hypoxia inducible factor 1 determine sufficiency of various metabolites, and in turn modulate cellular functions that includes control of endocytic membrane traffic. We also examine how certain metabolites can directly control endocytic traffic proteins, such as the regulation of specific protein glycosylation by limiting levels of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) produced by the hexosamine biosynthetic pathway. From these ideas emerge a growing appreciation that endocytic membrane traffic is orchestrated by many intrinsic signals derived from cell metabolism, allowing alignment of the functions of cell surface proteins with cellular metabolic requirements. Endocytic membrane traffic determines how cells interact with their environment, thus defining many aspects of nutrient uptake and energy consumption. We examine how intrinsic signals that reflect metabolic status of a cell regulate endocytic traffic of specific proteins, and, in some cases, exert broad control of endocytic membrane traffic phenomena. Hence, endocytic traffic is versatile and adaptable and can be modulated to meet the changing metabolic requirements of a cell.

Comparison of α2,6-sialyltransferases for sialylation of therapeutic proteins.

The development of therapeutic proteins for the treatment of numerous diseases is one of the fastest growing areas of biotechnology. Therapeutic efficacy and serum half-life are particularly important, and these properties rely heavily on the glycosylation state of the protein. Expression systems to produce authentically fully glycosylated therapeutic proteins with appropriate terminal sialic acids are not yet perfected. The in vitro modification of therapeutic proteins by recombinant sialyltransferases offers a promising and elegant strategy to overcome this problem. Thus, the detailed expression and characterization of sialyltransferases for completion of the glycan chains is of great interest to the community. We identified a novel α2,6-sialyltransferase from Helicobacter cetorum and compared it to the human ST6Gal1 and a Photobacterium sp. sialyltransferase using glycoprotein substrates in a 96-well microtiter-plate-based assay. We demonstrated that the recombinant α2,6-sialyltransferase from H. cetorum is an excellent catalyst for modification of N-linked glycans of different therapeutic proteins.

Directed evolution of bacterial polysialyltransferases.

Polysialyltransferases (polySTs) are glycosyltransferases that synthesize polymers of sialic acid found in vertebrates and some bacterial pathogens. Bacterial polySTs have utility in the modification of therapeutic proteins to improve serum half-life, and the potential for tissue engineering. PolySTs are membrane-associated proteins and as recombinant proteins suffer from inherently low solubility, low expression levels and poor thermal stability. To improve their physicochemical and biochemical properties, we applied a directed evolution approach using a FACS-based ultrahigh-throughput assay as a simple, robust and reliable screening method. We were able to enrich a large mutant library and, in combination with plate-based high-throughput secondary screening, we discovered mutants with increased enzymatic activity and improved stability compared to the wildtype enzyme. This work presents a powerful strategy for the screening of directed evolution libraries of bacterial polySTs to identify better catalysts for in vitro polysialylation of therapeutics.

Proteomic Analysis of the Secretome of Cellulomonas fimi ATCC 484 and Cellulomonas flavigena ATCC 482.

The bacteria in the genus Cellulomonas are known for their ability to degrade plant cell wall biomass. Cellulomonas fimi ATCC 484 and C. flavigena ATCC 482 have been the subject of much research into secreted cellulases and hemicellulases. Recently the genome sequences of both C. fimi ATCC 484 and C. flavigena ATCC 482 were published, and a genome comparison has revealed their full spectrum of possible carbohydrate-active enzymes (CAZymes). Using mass spectrometry, we have compared the proteins secreted by C. fimi and C. flavigena during growth on the soluble cellulose substrate, carboxymethylcellulose (CMC), as well as a soluble xylan fraction. Many known C. fimi CAZymes were detected, which validated our analysis, as were a number of new CAZymes and other proteins that, though identified in the genome, have not previously been observed in the secretome of either organism. Our data also shows that many of these are co-expressed on growth of either CMC or xylan. This analysis provides a new perspective on Cellulomonas enzymes and provides many new CAZyme targets for characterization.

Preparation of legionaminic acid analogs of sialo-glycoconjugates by means of mammalian sialyltransferases.

Legionaminic acids are analogs of sialic acid that occur in several bacteria. The most commonly occurring form is Leg5Ac7Ac, which differs from Neu5Ac only at the C7 (acetamido) and C9 (deoxy) positions. While these differences greatly reduce the susceptibility of Leg compounds to sialidases, several sialyltransferases have been identified that can use CMP-Leg5Ac7Ac as a donor (Watson et al. 2011). We report the successful modification with Leg5Ac7Ac of a glycolipid, GM1a, and two glycoproteins, interferon-α2b and α1-antitrypsin, by means of two mammalian sialyltransferases, namely porcine ST3Gal1 and human ST6Gal1. The Leg5Ac7Ac form of GD1a was not recognized by the myelin-associated glycoprotein (MAG, Siglec-4), confirming the importance of the glycerol moiety in the interaction of sialo-glycans with Siglecs.

Sialyltransferases with enhanced legionaminic acid transferase activity for the preparation of analogs of sialoglycoconjugates.

Legionaminic acids (Leg) are bacterial analogs of neuraminic acid, with the same stereochemistry but different substituents at C5, C7 and C9. Hence they may be incorporated into useful analogs of sialoglycoconjugates, and we previously reported two sialyltransferases that could utilize cytidine monophosphate (CMP)-Leg5Ac7Ac for preparation of Leg glycoconjugates, which were resistant to sialidases [Watson DC, Leclerc S, Wakarchuk WW, Young NM. 2011. Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid. Glycobiology. 21:99-108.]. These were the porcine ST3Gal1 and Pasteurella multocida sialyltransferases. We now report two additional sialyltransferases with superior Leg-transferase properties to the previous two. These are (i) a truncated form of a Photobacterium α2,6-sialyltransferase with an Ala-Met mutation in its active site, and (ii) an α2,3-sialyltransferase from Neisseria meningitidis MC58 with a higher transferase activity than the P. multocida enzyme, with either CMP-Neu5Ac or CMP-Leg5Ac7Ac as the donor. These enzymes will enable the production of useful Leg5Ac7Ac glycoconjugate derivatives with either α2,6 or α2,3 linkages and unique biological properties.

A glyco-gold nanoparticle based assay for α-2,8-polysialyltransferase from Neisseria meningitidis.

We designed a novel strategy for sensitively detecting the activity of α-2,8-polysialyltransferase (PST) by a combination of ganglioside GD3 functionalized gold nanoparticles and inactive endosialidase. We anticipate that this new method will facilitate the search for PST inhibitors as well as for improved mutant forms of PST in directed evolution experiments.

Biochemical characterization of a polysialyltransferase from Mannheimia haemolytica A2 and comparison to other bacterial polysialyltransferases.

Polysialic acids are bioactive carbohydrates found in eukaryotes and some bacterial pathogens. The bacterial polysialyltransferases (PSTs), which catalyze the synthesis of polysialic acid capsules, have previously been identified in select strains of Escherichia coli and Neisseria meningitidis and are classified in the Carbohydrate-Active enZYmes Database as glycosyltransferase family GT-38. In this study using DNA sequence analysis and functional characterization we have identified a novel polysialyltransferase from the bovine/ovine pathogen Mannheimia haemolytica A2 (PSTMh). The enzyme was expressed in recombinant form as a soluble maltose-binding-protein fusion in parallel with the related PSTs from E. coli K1 and N. meningitidis group B in order to perform a side-by-side comparison. Biochemical properties including solubility, acceptor preference, reaction pH optima, thermostability, kinetics, and product chain length for the enzymes were compared using a synthetic fluorescent acceptor molecule. PSTMh exhibited biochemical properties that make it an attractive candidate for chemi-enzymatic synthesis applications of polysialic acid. The activity of PSTMh was examined on a model glycoprotein and the surface of a neuroprogenitor cell line where the results supported its development for use in applications to therapeutic protein modification and cell surface glycan remodelling to enable cell migration at implantation sites to promote wound healing. The three PSTs examined here demonstrated different properties that would each be useful to therapeutic applications.

Review of phosphocholine substituents on bacterial pathogen glycans: synthesis, structures and interactions with host proteins.

Among the non-carbohydrate components of glycans, the addition of phosphocholine (ChoP) to the glycans of pathogens occurs more rarely than acetylation or methylation, but it has far more potent biological consequences. These arise from ChoP's multiple interactions with host proteins, which are important at all stages of the infection process. These stages include initial adherence to cells, encountering the host's innate immune system and then the adaptive immune system. Thus, in the initial stages of an infection, ChoP groups are an asset to the pathogen, but they can turn into a disadvantage subsequently. In this review, we have focussed on structural aspects of these phenomena. We describe the biosynthesis of the ChoP modification, the structures of the pathogen glycans known to carry ChoP groups and the host proteins that recognize ChoP.

Domain organization of the polymerizing mannosyltransferases involved in synthesis of the Escherichia coli O8 and O9a lipopolysaccharide O-antigens.

The Escherichia coli O9a and O8 polymannose O-polysaccharides (O-PSs) serve as model systems for the biosynthesis of bacterial polysaccharides by ATP-binding cassette transporter-dependent pathways. Both O-PSs contain a conserved primer-adaptor domain at the reducing terminus and a serotype-specific repeat unit domain. The repeat unit domain is polymerized by the serotype-specific WbdA mannosyltransferase. In serotype O9a, WbdA is a bifunctional α-(1→2)-, α-(1→3)-mannosyltransferase, and its counterpart in serotype O8 is trifunctional (α-(1→2), α-(1→3), and ß-(1→2)). Little is known about the detailed structures or mechanisms of action of the WbdA polymerases, and here we establish that they are multidomain enzymes. WbdA(O9a) contains two separable and functionally active domains, whereas WbdA(O8) possesses three. In WbdC(O9a) and WbdB(O9a), substitution of the first Glu of the EX(7)E motif had detrimental effects on the enzyme activity, whereas substitution of the second had no significant effect on activity in vivo. Mutation of the Glu residues in the EX(7)E motif of the N-terminal WbdA(O9a) domain resulted in WbdA variants unable to synthesize O-PS. In contrast, mutation of the Glu residues in the motif of the C-terminal WbdA(O9a) domain generated an enzyme capable of synthesizing an altered O-PS repeat unit consisting of only α-(1→2) linkages. In vitro assays with synthetic acceptors unequivocally confirmed that the N-terminal domain of WbdA(O9a) possesses α-(1→2)-mannosyltransferase activity. Together, these studies form a framework for detailed structure-function studies on individual domains and a strategy applicable for dissection and analysis of other multidomain glycosyltransferases.

Biosynthesis of the polymannose lipopolysaccharide O-antigens from Escherichia coli serotypes O8 and O9a requires a unique combination of single- and multiple-active site mannosyltransferases.

The Escherichia coli O9a and O8 O-antigen serotypes represent model systems for the ABC transporter-dependent synthesis of bacterial polysaccharides. The O9a and O8 antigens are linear mannose homopolymers containing conserved reducing termini (the primer-adaptor), a serotype-specific repeat unit domain, and a terminator. Synthesis of these glycans occurs on the polyisoprenoid lipid-linked primer, undecaprenol pyrophosphoryl-GlcpNAc, by two conserved mannosyltransferases, WbdC and WbdB, and a serotype-specific mannosyltransferase, WbdA. The glycan structure and pattern of conservation in the O9a and O8 mannosyltransferases are not consistent with the existing model of O9a biosynthesis. Here we establish a revised pathway using a combination of in vivo (mutant complementation) experiments and in vitro strategies with purified enzymes and synthetic acceptors. WbdC and WbdB synthesize the adaptor region, where they transfer one and two α-(1→3)-linked mannose residues, respectively. The WbdA enzymes are solely responsible for forming the repeat unit domains of these O-antigens. WbdA(O9a) has two predicted active sites and polymerizes a tetrasaccharide repeat unit containing two α-(1→3)- and two α-(1→2)-linked mannopyranose residues. In contrast, WbdA(O8) polymerizes trisaccharide repeat units containing single α-(1→3)-, α-(1→2)-, and ß-(1→2)-mannopyranoses. These studies illustrate assembly systems exploiting several mannosyltransferases with flexible active sites, arranged in single- and multiple-domain formats.