Design space determination to optimize DNA complexation and full capsid formation in transient rAAV manufacturing.

Recombinant adeno-associated virus (rAAV) vectors are a promising platform for in vivo gene therapies. However, cost-effective, well-characterized processes necessary to manufacture rAAV therapeutics are challenging to develop without an understanding of how process parameters (PPs) affect rAAV product quality attributes (PQAs). In this work, a central composite orthogonal experimental design was employed to examine the influence of four PPs for transient transfection complex formation (polyethylenimine:DNA [PEI:DNA] ratio, total DNA/cell, cocktail volume, and incubation time) on three rAAV PQAs related to capsid content (vector genome titer, vector genome:capsid particle ratio, and two-dimensional vector genome titer ratio). A regression model was established for each PQA using partial least squares, and a design space (DS) was defined in which Monte Carlo simulations predicted < 1% probability of failure (POF) to meet predetermined PQA specifications. Of the three PQAs, viral genome titer was most strongly correlated with changes in complexation PPs. The DS and acceptable PP ranges were largest when incubation time and cocktail volume were kept at mid-high setpoints, and PEI:DNA ratio and total DNA/cell were at low-mid setpoints. Verification experiments confirmed model predictive capability, and this work establishes a framework for studying other rAAV PPs and their relationship to PQAs.

Advancing a rapid, high throughput screening platform for optimization of lentivirus production.

BACKGROUND: Lentiviral vectors (LVVs) hold great promise as delivery tools for gene therapy and chimeric antigen receptor T cell (CAR-T) therapy. Their ability to target difficult to transfect cells and deliver genetic payloads that integrate into the host genome makes them ideal delivery candidates. However, several challenges remain to be addressed before LVVs are more widely used as therapeutics including low viral vector concentrations and the absence of suitable scale-up methods for large-scale production. To address these challenges, we have developed a high throughput microscale HEK293 suspension culture platform that enables rapid screening of conditions for improving LVV productivity. KEY RESULTS: High density culture (40 million cells mL-1 ) of HEK293 suspension cells in commercially available media was achieved in microscale 96-deep well plate platform at liquid volumes of 200 µL. Comparable transfection and LVV production efficiencies were observed at the microscale, in conventional shake flasks and a 1-L bioreactor, indicating that significant scale-down does not affect LVV concentrations and predictivity of scale-up. Optimization of production step allowed for final yields of LVVs to reach 1.5 × 107  TU mL-1 . CONCLUSIONS: The ability to test a large number of conditions simultaneously with minimal reagent use allows for the rapid optimization of LVV production in HEK293 suspension cells. Therefore, such a system may serve as a valuable tool in early stage process development and can be used as a screening tool to improve LVV concentrations for both batch and perfusion based systems.

Development and Optimization of a Hydrophobic Interaction Chromatography-Based Method of AAV Harvest, Capture, and Recovery.

With many ongoing clinical trials utilizing adeno-associated virus (AAV) gene therapy, it is necessary to find scalable and serotype-independent primary capture and recovery methods to allow for efficient and robust manufacturing processes. Here, we demonstrate the ability of a hydrophobic interaction chromatography membrane to capture and recover AAV1, AAV5, AAV8, and AAV "Mutant C" (a novel serotype incorporating elements of AAV3B and AAV8) particles from cell culture media and cell lysate with recoveries of 76%-100% of loaded material, depending on serotype. A simple, novel technique that integrates release and recovery of cell-associated AAV capsids is demonstrated. We show that by the addition of lyotropic salts to AAV-containing cell suspensions, AAV is released at an equivalent efficiency to mechanical lysis. The addition of the lyotropic salt also promotes a phase separation, which allows physical removal of large amounts of DNA and insoluble cellular debris from the AAV-containing aqueous fraction. The AAV is then captured and eluted from a hydrophobic interaction chromatography membrane. This integrated lysis and primary capture and recovery technique facilitates substantial removal of host-cell DNA and host-cell protein impurities.

Affinity recovery of lentivirus by diaminopelargonic acid mediated desthiobiotin labelling.

Desthiobiotin-tagged lentiviral vectors have been metabolically produced by DBL producer cells in a 7,8-diaminopelargonic acid (7-DAPA) dependent manner for envelope independent, single-step affinity purification. 7-DAPA, which has little or no affinity for avidin/streptavidin, was synthesised and verified by NMR spectroscopy and mass spectrometry. By expressing the biotin acceptor, biotin ligase and desthiobiotin synthase bioD, DBL cells converted exogenous 7-DAPA into membrane-bound desthiobiotin. Desthiobiotin on the DBL cell surface was visualised by confocal microscopy and the desthiobiotin density was quantified by HABA-avidin assay. Desthiobiotin was then spontaneously incorporated onto the surface of lentiviral vectors produced by the DBL cells. It has been demonstrated by flow cytometry that the desthiobiotinylated lentiviruses were captured from the crude 7-DAPA-containing viral supernatant by Streptavidin Magnespheres and eluted by biotin solution efficiently whilst retaining infectivity. The practical, high yielding virus purification using Pierce monomeric avidin coated columns indicates a highly efficient biotin-dependent recovery of infectious lentiviruses at 68%. The recovered lentiviral vectors had a high purity and the majority were eluted within 45 min. This 7-DAPA mediated desthiobiotinylation technology can be applied in scalable production of viral vectors for clinical gene therapy.

Arctic permafrost active layer detachments stimulate microbial activity and degradation of soil organic matter.

Large quantities of soil organic carbon in Arctic permafrost zones are becoming increasingly unstable due to a warming climate. High temperatures and substantial rainfall in July 2007 in the Canadian High Arctic resulted in permafrost active layer detachments (ALDs) that redistributed soils throughout a small watershed in Nunavut, Canada. Molecular biomarkers and NMR spectroscopy were used to measure how ALDs may lead to microbial activity and decomposition of previously unavailable soil organic matter (SOM). Increased concentrations of extracted bacterial phospholipid fatty acids (PLFAs) and large contributions from bacterial protein/peptides in the NMR spectra at recent ALDs suggest increased microbial activity. PLFAs were appreciably depleted in a soil sample where ALDs occurred prior to 2003. However an enrichment of bacterial derived peptidoglycan was observed by (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) and (1)H diffusion edited (DE) NMR and enhanced SOM degradation was observed by (13)C solid-state NMR. These data suggest that a previous rise in microbial activity, as is currently underway at the recent ALD site, led to degradation and depletion of labile SOM components. Therefore, this study indicates that ALDs may amplify climate change due to the release of labile SOM substrates from thawing High Arctic permafrost.

The application of NMR spectroscopy to functional glycomics.

Glycomics which is the study of saccharides and genes responsible for their formation requires the continuous development of rapid and sensitive methods for the identification of glycan structures. It involves glycoanalysis which relies upon the development of methods for determining the structure and interactions of carbohydrates. For the application of functional glycomics to microbial virulence, carbohydrates and their associated metabolic and carbohydrate processing enzymes and respective genes can be identified and exploited as targets for drug discovery, glyco-engineering, vaccine design, and detection and diagnosis of diseases. Glycomics also encompasses the detailed understanding of carbohydrate-protein interactions and this knowledge can be applied to research efforts focused toward the development of vaccines and immunological therapies to alleviate infectious diseases.

N-linked protein glycosylation in a bacterial system.

N-Linked protein glycosylation is conserved throughout the three domains of life and influences protein function, stability, and protein complex formation. N-Linked glycosylation is an essential process in Eukaryotes; however, although N-glycosylation affects multiple cellular processes in Archaea and Bacteria, it is not needed for cell survival. Methods for the analyses of N-glycosylation in eukaryotes are well established, but comparable techniques for the analyses of the pathways in Bacteria and Archaea are needed. In this chapter we describe new methods for the detection and analyses of N-linked, and the recently discovered free oligosaccharides (fOS), from whole cell lysates of Campylobacter jejuni using non-specific pronase E digestion and permethylation followed by mass spectrometry. We also describe the expression and immunodetection of the model N-glycoprotein, AcrA, fused to a hexa-histidine tag to follow protein glycosylation in C. jejuni. This chapter concludes with the recent demonstration that high-resolution magic angle spinning NMR of intact bacterial cells provides a rapid, non-invasive method for analyzing fOS in C. jejuni in vivo. This combination of techniques provides a powerful tool for the exploration, quantification, and structural analyses of N-linked and free oligosaccharides in the bacterial system.

Study of free oligosaccharides derived from the bacterial N-glycosylation pathway.

The food-borne pathogen Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis worldwide and the most frequent antecedent in neuropathies such as the Guillain-Barré and Miller Fisher syndromes. C. jejuni was demonstrated to possess an N-linked protein glycosylation pathway that adds a conserved heptasaccharide to >40 periplasmic and membrane proteins. Recently, we showed that C. jejuni also produces free heptasaccharides derived from the N-glycan pathway reminiscent of the free oligosaccharides (fOS) produced by eukaryotes. Herein, we demonstrate that C. jejuni fOS are produced in response to changes in the osmolarity of the environment and bacterial growth phase. We provide evidence showing the conserved WWDYG motif of the oligosaccharyltransferase, PglB, is necessary for fOS release into the periplasm. This report demonstrates that fOS from an N-glycosylation pathway in bacteria are potentially equivalent to osmoregulated periplasmic glucans in other Gram-negative organisms.

Bioactivation of minocycline to reactive intermediates by myeloperoxidase, horseradish peroxidase, and hepatic microsomes: implications for minocycline-induced lupus and hepatitis.

Of the tetracyclines, minocycline is unique in causing a significant incidence of a lupus-like syndrome and autoimmune hepatitis. It is also unique among the tetracyclines in having a para-N,N-dimethylaminophenol ring. Many drugs that cause autoimmune reactions are oxidized to reactive metabolites by the myeloperoxidase (MPO) system of macrophages. In this study, we showed that minocycline is oxidized to reactive intermediates by MPO/H(2)O(2)/Cl(-), HOCl, horseradish peroxidase/H(2)O(2), or hepatic microsomes. When trapped with N-acetylcysteine (NAC), two adducts with protonated molecular ions at m/z 619 were isolated and analyzed by NMR. One represents attack of the aromatic D ring by NAC meta to the N,N-dimethylamino group, which implies that the reactive intermediate was a quinone iminium ion. The NMR of the other adduct, which was not observed when minocycline was oxidized by hepatic microsomes, indicates that the NAC is attached at the junction of the B and C rings. In the oxidation by HOCl, we found an intermediate with a protonated molecular ion of m/z 510 that represents the addition of HOCl to minocycline. The HOCl presumably adds across the double bond of the B ring, and reaction of this intermediate with NAC led to the second NAC adduct. We were surprised to find that the same NAC adduct was not observed after oxidation of tetracycline with HOCl, even though this part of the tetracycline structure is the same as for minocycline. We propose that one or more of these reactive metabolites are responsible for the idiosyncratic drug reactions that are specific to this tetracycline.

The structural basis for catalytic function of GMD and RMD, two closely related enzymes from the GDP-D-rhamnose biosynthesis pathway.

The rare 6-deoxysugar D-rhamnose is a component of bacterial cell surface glycans, including the D-rhamnose homopolymer produced by Pseudomonas aeruginosa, called A-band O polysaccharide. GDP-D-rhamnose synthesis from GDP-D-mannose is catalyzed by two enzymes. The first is a GDP-D-mannose-4,6-dehydratase (GMD). The second enzyme, RMD, reduces the GMD product (GDP-6-deoxy-D-lyxo-hexos-4-ulose) to GDP-d-rhamnose. Genes encoding GMD and RMD are present in P. aeruginosa, and genetic evidence indicates they act in A-band O-polysaccharide biosynthesis. Details of their enzyme functions have not, however, been previously elucidated. We aimed to characterize these enzymes biochemically, and to determine the structure of RMD to better understand what determines substrate specificity and catalytic activity in these enzymes. We used capillary electrophoresis and NMR analysis of reaction products to precisely define P. aeruginosa GMD and RMD functions. P. aeruginosa GMD is bifunctional, and can catalyze both GDP-d-mannose 4,6-dehydration and the subsequent reduction reaction to produce GDP-D-rhamnose. RMD catalyzes the stereospecific reduction of GDP-6-deoxy-D-lyxo-hexos-4-ulose, as predicted. Reconstitution of GDP-D-rhamnose biosynthesis in vitro revealed that the P. aeruginosa pathway may be regulated by feedback inhibition in the cell. We determined the structure of RMD from Aneurinibacillus thermoaerophilus at 1.8 A resolution. The structure of A. thermoaerophilus RMD is remarkably similar to that of P. aeruginosa GMD, which explains why P. aeruginosa GMD is also able to catalyze the RMD reaction. Comparison of the active sites and amino acid sequences suggests that a conserved amino acid side chain (Arg185 in P. aeruginosa GMD) may be crucial for orienting substrate and cofactor in GMD enzymes.

Characterization of WbpB, WbpE, and WbpD and reconstitution of a pathway for the biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic acid in Pseudomonas aeruginosa.

The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an unusual sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA). wbpB, wbpE, and wbpD are thought to encode oxidase, transaminase, and N-acetyltransferase enzymes. To characterize their functions, recombinant proteins were overexpressed and purified from heterologous hosts. Activities of His(6)-WbpB and His(6)-WbpE were detected only when both proteins were combined in the same reaction. Using a direct MALDI-TOF mass spectrometry approach, we identified ions that corresponded to the predicted products of WbpB (UDP-3-keto-d-GlcNAcA) and WbpE (UDP-d-GlcNAc3NA) in the coupled enzyme-substrate reaction. Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent with the expected product of WbpD (UDP-d-GlcNAc3NAcA) was identified. Preparative quantities of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA were enzymatically synthesized. These compounds were purified by high-performance liquid chromatography, and their structures were elucidated by NMR spectroscopy. This is the first report of the functional characterization of these proteins, and the enzymatic synthesis of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA.

Flagellar glycosylation in Clostridium botulinum.

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.

Flagellin glycosylation in Pseudomonas aeruginosa PAK requires the O-antigen biosynthesis enzyme WbpO.

Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.

Commonality and biosynthesis of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni.

In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.

In silico identification of functional divergence between the multiple groEL gene paralogs in Chlamydiae.

BACKGROUND: Heat-shock proteins are specialized molecules performing different and essential roles in the cell including protein degradation, folding and trafficking. GroEL is a 60 Kda heat-shock protein ubiquitous in bacteria and has been regarded as an important molecule implicated in chronic inflammatory processes caused by Chlamydiae infections. GroEL in Chlamydiae became duplicated at the origin of the Chlamydiae lineage presenting three distinct molecular chaperones, namely the original protein GroEL1 (Ct110), and its paralogous proteins GroEL2 (Ct604) and GroEL3 (Ct755). These chaperones present differential and independent expressions during the different stages of Chlamydiae infections and have been suggested to present differential physiological and regulatory roles. RESULTS: In this comprehensive in silico study we show that GroEL protein paralogs have diverged functionally after the different gene duplication events and that this divergence has occurred mainly between GroEL3 and GroEL1. GroEL2 presents an intermediate functional divergence pattern from GroEL1. Our results point to the different protein-protein interaction patterns between GroEL paralogs and known GroEL protein clients supporting their functional divergence after groEL gene duplication. Analysis of selective constraints identifies periods of adaptive evolution after gene duplication that led to the fixation of amino acid replacements in GroEL protein domains involved in the interaction with GroEL protein clients. CONCLUSION: We demonstrate that GroEL protein copies in Chlamydiae species have diverged functionally after the gene duplication events. We also show that functional divergence has occurred in important functional regions of these GroEL proteins and that very probably have affected the ancestral GroEL regulatory role and protein-protein interaction patterns with GroEL client proteins. Most of the amino acid replacements that have affected interaction with protein clients and that were responsible for the functional divergence between GroEL paralogs were fixed by adaptive evolution after the groEL gene duplication events.

Targeted metabolomics analysis of Campylobacter coli VC167 reveals legionaminic acid derivatives as novel flagellar glycans.

Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic acid biosynthetic enzymes. This is the first report of legionaminic acid in Campylobacter sp. and the first report of legionaminic acid derivatives as modifications on a protein.

Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases from respiratory pathogens.

The heteropolymeric O-antigen of the lipopolysaccharide from Pseudomonas aeruginosa serogroup O5 as well as the band-A trisaccharide from Bordetella pertussis contain the di-N-acetylated mannosaminuronic acid derivative, beta-D-ManNAc3NAcA (2,3-diacetamido-2,3-dideoxy-beta-D-mannuronic acid). The biosynthesis of the precursor for this sugar is proposed to require five steps, through which UDP-alpha-D-GlcNAc (UDP-N-acetyl-alpha-D-glucosamine) is converted via four steps into UDP-alpha-D-GlcNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid), and this intermediate compound is then epimerized by WbpI (P. aeruginosa), or by its orthologue, WlbD (B. pertussis), to form UDP-alpha-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-mannuronic acid). UDP-alpha-D-GlcNAc3NAcA, the proposed substrate for WbpI and WlbD, was obtained through chemical synthesis. His6-WbpI and His6-WlbD were overexpressed and then purified by affinity chromatography using FPLC. Capillary electrophoresis was used to analyse reactions with each enzyme, and revealed that both enzymes used UDP-alpha-D-GlcNAc3NAcA as a substrate, and reacted optimally in sodium phosphate buffer (pH 6.0). Neither enzyme utilized UDP-alpha-D-GlcNAc, UDP-alpha-D-GlcNAcA (UDP-2-acetamido-2,3-dideoxy-alpha-D-glucuronic acid) or UDP-alpha-D-GlcNAc3NAc (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucose) as substrates. His6-WbpI or His6-WlbD reactions with UDP-alpha-D-GlcNAc3NAcA produce a novel peak with an identical retention time, as shown by capillary electrophoresis. To unambiguously characterize the reaction product, enzyme-substrate reactions were allowed to proceed directly in the NMR tube and conversion of substrate into product was monitored over time through the acquisition of a proton spectrum at regular intervals. Data collected from one- and two-dimensional NMR experiments showed that His6-WbpI catalysed the 2-epimerization of UDP-alpha-D-GlcNAc3NAcA, converting it into UDP-alpha-D-ManNAc3NAcA. Collectively, these results provide evidence that WbpI and WlbD are UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases.