Efficient One-Pot Synthesis of Uridine Diphosphate Galactose Employing a Trienzyme System.

The limited availability of high-cost nucleotide sugars is a significant constraint on the application of their downstream products (glycosides and prebiotics) in the food or pharmaceutical industry. To better solve the problem, this study presented a one-pot approach for the biosynthesis of UDP-Gal using a thermophilic multienzyme system consisting of GalK, UGPase, and PPase. Under optimal conditions, a 2 h reaction resulted in a UTP conversion rate of 87.4%. In a fed-batch reaction with Gal/ATP = 20 mM:10 mM, UDP-Gal accumulated to 33.76 mM with a space-time yield (STY) of 6.36 g/L·h-1 after the second feeding. In repetitive batch synthesis, the average yield of UDP-Gal over 8 cycles reached 10.80 g/L with a very low biocatalyst loading of 0.002 genzymes/gproduct. Interestingly, Galk (Tth0595) could synthesize Gal-1P using ADP as a donor of phosphate groups, which had never been reported before. This approach possessed the benefits of high synthesis efficiency, low cost, and superior reaction system stability, and it provided new insights into the rapid one-pot synthesis of UDP-Gal and high-value glycosidic compounds.

Cell-Free Multi-Enzyme Synthesis and Purification of Uridine Diphosphate Galactose.

High costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for the scalable, biocatalytic synthesis and purification of UDP-galactose. UDP-galactose was produced with a titer of 48 mM (27.2 g/L) in a small-scale batch process (200 µL) within 24 h using 0.02 genzyme /gproduct . Through in-situ ATP regeneration, the amount of ATP (0.6 mM) supplemented was around 240-fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP-galactose with a purity of 92 %. The synthesis was transferred to 1 L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4 g/L UDP-galactose were produced within 23 h with a synthesis yield of 71 % and a biocatalyst load of 0.05 gtotal_protein /gproduct . The costs for substrates per gram of UDP-galactose synthesized were around 0.26 €/g.

The PGRMC1 Antagonist AG-205 Inhibits Synthesis of Galactosylceramide and Sulfatide.

Sulfatide synthesis in the human renal cancer cell line SMKT-R3 was strongly inhibited in the presence of low µM concentrations of AG-205, a progesterone receptor membrane component 1 (PGRMC1) antagonist. This was also the case in Chinese hamster ovary (CHO) cells stably transfected with UDP-galactose: ceramide galactosyltransferase and cerebroside sulfotransferase, the two enzymes required for sulfatide synthesis. In CHO cells synthesizing galactosylceramide but not sulfatide, galactosylceramide was also strongly reduced, suggesting an effect at the level of galactolipid synthesis. Notably, AG-205 inhibited galactosylceramide synthesis to a similar extent in wild type CHO cells and cells that lack PGRMC1 and/or PGRMC2. In vitro enzyme activity assays showed that AG-205 is an inhibitor of UDP-galactose: ceramide galactosyltransferase, but not cerebroside sulfotransferase. This study shows that PGRMC1 is only one of several targets of AG-205 and should be used with caution, especially in studies using cells synthesizing galactosylceramide and sulfatide.

Identification of novel potential interaction partners of UDP-galactose (SLC35A2), UDP-N-acetylglucosamine (SLC35A3) and an orphan (SLC35A4) nucleotide sugar transporters.

Nucleotide sugar transporters (NSTs) are ER and Golgi-resident members of the solute carrier 35 (SLC35) family which supply substrates for glycosylation by exchanging lumenal nucleotide monophosphates for cytosolic nucleotide sugars. Defective NSTs have been associated with congenital disorders of glycosylation (CDG), however, molecular basis of many types of CDG remains poorly characterized. To better understand the biology of NSTs, we identified potential interaction partners of UDP-galactose transporter (SLC35A2), UDP-N-acetylglucosamine transporter (SLC35A3) and an orphan nucleotide sugar transporter SLC35A4 of to date unassigned specificity. For this purpose, each of the SLC35A2-A4 proteins was used as a bait in four independent pull-down experiments and the identity of the immunoprecipitated material was discovered using MS techniques. From the candidate list obtained, we selected a few for which the interaction was confirmed in vitro using the NanoBiT system, a split luciferase-based luminescent technique. NSTs have been shown to interact with two ATPases (ATP2A2, ATP2C1), Golgi pH regulator B (GPR89B) and calcium channel (TMCO1), which may reflect the regulation of glycosylation by ion homeostasis, and with basigin (BSG). Our findings provide a starting point for the NST interaction network discovery in order to better understand how glycosylation is regulated and linked to other cellular processes. SIGNIFICANCE: Despite the facts that nucleotide sugar transporters are a key component of the protein glycosylation machinery, and deficiencies in their activity underlie serious metabolic diseases, biology, function and regulation of these essential proteins remain enigmatic. In this study we have advanced the field by identifying sets of new potential interaction partners for UDP-galactose transporter (SLC35A2), UDP-N-acetylglucosamine transporter (SLC35A3) and an orphan transporter SLC35A4 of yet undefined role. Several of these new interactions were additionally confirmed in vitro using the NanoBiT system, a split luciferase complementation assay. This work is also significant in that it addresses the overall challenge of discovering membrane protein interaction partners by a detailed comparison of 4 different co-immunoprecipitation strategies and by custom sample preparation and data processing workflows.

UPLC-MS based plasma metabolomics and lipidomics reveal alterations associated with IgG4-related disease.

OBJECTIVE: The pathogenesis of IgG4-related disease (IgG4-RD) remains unclear. Metabolomic profiling of IgG4-RD patients offers an opportunity to identify novel pathophysiological targets and biomarkers. This study aims to identify potential plasma biomarkers associated with IgG4-RD. METHODS: Thirty newly diagnosed IgG4-RD patients, age-matched healthy controls and post-treated IgG4-RD patients were enrolled. Patients' clinical data, laboratory parameters and plasma were collected. Plasma was measured for ultraperformance liquid chromatography-tandem mass spectrometry based metabolomics and lipidomics profiling. Multivariate and univariate statistical analyses were conducted to identify potential biomarkers. The receiver operating characteristic and the correlations between biomarkers and clinical parameters were investigated. RESULTS: The plasma metabolites are altered among healthy controls, newly diagnosed IgG4-RD and post-treated IgG4-RD groups. Of the identified features, eight metabolites were significantly perturbed in the IgG4-RD group, including glyceric acid 1,3-biphosphate (1,3-BPG), uridine triphosphate (UTP), uridine diphosphate glucose (UDP-Glc) or uridine diphosphate galactose (UDP-Gal), lysophospholipids, linoleic acid derivatives and ceramides. Receiver operating characteristic analysis indicated that UTP, UDP-Glc/UDP-Gal and LysoPC (18:1) had high sensitivity and specificity in diagnosis of IgG4-RD. A Pearson correlation analysis showed that 1,3-BPG and UTP were strongly correlated with clinical parameters. CONCLUSION: IgG4-RD patients have a unique plasma metabolomic profile compared with healthy controls. Our study suggested that metabolomic profiling may provide important insights into pathophysiology and testable biomarkers for diagnosis of IgG4-RD.

How inverting ß-1,4-galactosyltransferase-1 can quench a high charge of the by-product UDP3- in catalysis: a QM/MM study of enzymatic reaction with native and UDP-5' thio galactose substrates.

The catalysis of inverting glycosyltransferases consists of several biophysical and biochemical processes during which the transfer of a sugar residue from the purine phosphate donor substrate to an acceptor substrate occurs with stereo-inversion of the anomeric C1 center at a product. During catalysis a highly charged phosphate by-product (UDP3-) is formed and a mechanism of how the enzyme stabilizes it back to the UDP2- form is not known. Using methods of molecular modeling (hybrid DFT-QM/MM calculations) we proposed and validated a catalytic mechanism of bovine inverting ß-1,4-galactosyltransferase-1 (ß4Gal-T1) with native (UDP-galactose) and thio donor substrates (UDP-5' thio galactose). We focused on three aspects of the mechanism not yet investigated: (i) the formation of an oxocarbenium ion intermediate, which was only found for the retaining glycosyltransferases for the time being; (ii) the mechanism of stabilization of a highly charged phosphate by-product (UDP3-) back to its standard in vivo form (UDP2-); (iii) explanation for why in experimental measurements the rate of catalysis with the thio donor substrate is only 8% of the rate of that with the natural substrate. To understand the differences in the interaction patterns between the complexes enzyme : UDP-Gal and enzyme : UDP-5S-Gal, fragmented molecular orbital (FMO) decomposition energy analysis was carried out at the DFT level.

Clickable Galactose Analogues for Imaging Glycans in Developing Zebrafish.

Galactose is one of only nine monosaccharide precursors used to build complex glycans in vertebrates. Defects in galactose metabolism cause galactosemia and lysosomal storage diseases, and the ability to visualize metabolic flux through these pathways would help to understand mechanisms underlying disease pathogenesis. Bioorthogonal metabolic reporters are widely used tools to image glycan biosynthesis; however, to date, no galactose analogues have capitalized on this strategy. We demonstrate that the galactose salvage pathway is remarkably intolerant of unnatural galactose and galactose-1-phosphate analogues. Subtle modifications to uridine diphosphate galactose (UDP-Gal), which is the universal donor for galactosyltransferases, however, yielded effective metabolic probes for labeling glycans in vivo. We applied 6-alkynyl UDP-Gal, followed by click chemistry tagging, to visualize glycosylation during zebrafish development, revealing a striking accumulation into glycan-rich ridges within the organism's enveloping layer. UDP-Gal analogues represent a new class of glycan metabolic probes for revealing physiological and pathological changes in glycosylation in vivo.

Expression, purification, and characterization of a new Glucosyltransferase involved in the third step of O-antigen repeating-unit biosynthesis of Escherichia coli O152.

The O antigen is indispensable for the full function and virulence of pathogenic bacteria. During O-repeating unit (RU) biosynthesis, committed glycosyltransferases (GTs) transfer various sugars from an activated sugar donor to the appropriate lipid carrier sequentially. While the nucleotide sequence specific for O antigen of pathogenic bacteria is already known, the exact substrate specificity of most hypothetical GTs have yet be characterized. In the present paper, we report the biochemical characterization of one alpha-glucosyltransferase, WfgE, a member of GT family 4. This enzyme is implicated in the pentasaccharide RU biosynthetic pathway of E. coli O152 O antigen. A chemoenzymatically synthesized acceptor (GlcGlcNAc α-PP-O(CH2)10CH3) was used to characterize the WfgE activity. The enzyme product was determined to have a 1,2-linkage using strategy based on collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MSn). The lack of a DxD motif and its high activity without divalent metal ions suggests that WfgE belongs to the GT-B fold superfamily. The enzyme is specific for beta-glucose or galactose-terminating acceptor substrates, and in particular UDP-glucose but also UDP-galactose as donor substrates. Our results suggest that WfgE catalyses the addition of the third sugar residue of the E. coli O152 O-RU. The recombinant GST-WfgE was solubilized and further purified to homogeneity via GST affinity chromatography, paving the way for structure-function relationship studies.

cDNA isolation and functional characterization of UDP-glucose 4-epimerase from Davallia divaricate.

UDP-glucose 4-epimerase (UGE) is a universal enzyme responsible for interconversion of UDP-glucose and UDP-galactose. However, the gene encoding UGE from Davallia divaricate is elusive. In this study, two UGE genes, ddUGE1 and ddUGE2, were isolated and cloned from D. divaricate using a transcriptome-guided search strategy. Two unigenes sharing high sequence identity with UGE homologous genes were selected from transcriptome assembly. The enzymes, further functionally expressed in Escherichia coli, exhibit narrow substrate specificity. The biochemical characterization assays of DdUGE1 and DdUGE2 showed good thermal and pH stability, and metal ion independence, which provides a meaningful feature for biotechnological applications.[Formula: see text].

Functional Characterization and Structural Basis of an Efficient Di-C-glycosyltransferase from Glycyrrhiza glabra.

A highly efficient di-C-glycosyltransferase GgCGT was discovered from the medicinal plant Glycyrrhiza glabra. GgCGT catalyzes a two-step di-C-glycosylation of flopropione-containing substrates with conversion rates of >98%. To elucidate the catalytic mechanisms of GgCGT, we solved its crystal structures in complex with UDP-Glc, UDP-Gal, UDP/phloretin, and UDP/nothofagin, respectively. Structural analysis revealed that the sugar donor selectivity was controlled by the hydrogen-bond interactions of sugar hydroxyl groups with D390 and other key residues. The di-C-glycosylation capability of GgCGT was attributed to a spacious substrate-binding tunnel, and the G389K mutation could switch di- to mono-C-glycosylation. GgCGT is the first di-C-glycosyltransferase with a crystal structure, and the first C-glycosyltransferase with a complex structure containing a sugar acceptor. This work could benefit the development of efficient biocatalysts to synthesize C-glycosides with medicinal potential.

Designer biocatalysts for direct incorporation of exogenous galactose into globotriose.

Galactose is ubiquitous. The synthesis of galactose-containing oligosaccharides using Leloir galactosyltransferase requires uridine diphosphate (UDP)-galactose as the precursor. Of all UDP-galactose synthesis pathways developed for in vitro synthesis, the salvage pathway represents the simplest route. In this study, for the first time, we designed and constructed an Escherichia coli strain to use salvage pathway for UDP-galactose synthesis, demonstrating effective and direct incorporation of exogenous galactose into globotriose (Gb3). Successful establishment of salvage pathway enabled a complete delineation of carbon and energy source. Consequently, the designed biocatalyst was able to achieve high yield synthesis from galactose (0.95 moles of Gb3/moles galactose consumed) and a high product titer (2 g/L) in shaker flask within 24 hr. Elimination of limitation in acceptor sugar via homologous overexpression of LacY, the transporter for lactose, further improved the synthesis, raising Gb3 titer to 6 g/L in 24 hr and 7.5 g/L in 48 hr. The design principles successfully demonstrated in this study could be broadly applied for synthesis of other galactose-containing oligosaccharides. This study also illustrates a valid strategy to overcome limitation in the transport of acceptor sugar. As lactose is one of the most important basal structures, the significant improvement in synthesis through its enhanced transport could be emulated in numerous other lactose-based oligosaccharides.

Suppression of Alternative Lipooligosaccharide Glycosyltransferase Activity by UDP-Galactose Epimerase Enhances Murine Lung Infection and Evasion of Serum IgM.

In pathogens that produce lipooligosaccharide (LOS), sugar residues within the surface-exposed LOS outer core mediate interactions with components of the host immune system, promoting bacterial infection. Many LOS structures are controlled by phase variation mediated by random slipped-strand base mispairing, which can reversibly switch gene expression on or off. Phase variation diversifies the LOS, however its adaptive role is not well-understood. Nontypeable Haemophilus influenzae (NTHi) is an important pathogen that causes a range of illnesses in the upper and lower respiratory tract. In NTHi a phase variable galactosyltransferase encoded by lic2A initiates galactose chain extension of the LOS outer core. The donor substrate for Lic2A, UDP-galactose, is generated from UDP-glucose by UDP-galactose epimerase encoded by galE. Our previous fitness profiling of H. influenzae mutants in a murine lung model showed that the galE mutant had a severe survival defect, while the lic2A mutant's defect was modest, leading us to postulate that unidentified factors act as suppressors of potential defects in a lic2A mutant. Herein we conducted a genome-wide genetic interaction screen to identify genes epistatic on lic2A for survival in the murine lung. An unexpected finding was that galE mutants exhibited restored virulence properties in a lic2A mutant background. We identified an alternative antibody epitope generated by Lic2A in the galE mutant that increased sensitivity to classical complement mediated killing in human serum. Deletion of lic2A or restoration of UDP-galactose synthesis alleviated the galE mutant's virulence defects. These studies indicate that when deprived of its galactosyl substrate, Lic2A acquires an alternative activity leading to increased recognition of NTHi by IgM and decreased survival in the lung model. Biofilm formation was increased by deletion of galE and by increased availability of UDP-GlcNAc precursors that can compete with UDP-galactose production. NTHi's ability to reversibly inactivate lic2A by phase-variation may influence survival in niches of infection in which UDP-Galactose levels are limiting.

The Metabolic Map into the Pathomechanism and Treatment of PGM1-CDG.

Phosphoglucomutase 1 (PGM1) encodes the metabolic enzyme that interconverts glucose-6-P and glucose-1-P. Mutations in PGM1 cause impairment in glycogen metabolism and glycosylation, the latter manifesting as a congenital disorder of glycosylation (CDG). This unique metabolic defect leads to abnormal N-glycan synthesis in the endoplasmic reticulum (ER) and the Golgi apparatus (GA). On the basis of the decreased galactosylation in glycan chains, galactose was administered to individuals with PGM1-CDG and was shown to markedly reverse most disease-related laboratory abnormalities. The disease and treatment mechanisms, however, have remained largely elusive. Here, we confirm the clinical benefit of galactose supplementation in PGM1-CDG-affected individuals and obtain significant insights into the functional and biochemical regulation of glycosylation. We report here that, by using tracer-based metabolomics, we found that galactose treatment of PGM1-CDG fibroblasts metabolically re-wires their sugar metabolism, and as such replenishes the depleted levels of galactose-1-P, as well as the levels of UDP-glucose and UDP-galactose, the nucleotide sugars that are required for ER- and GA-linked glycosylation, respectively. To this end, we further show that the galactose in UDP-galactose is incorporated into mature, de novo glycans. Our results also allude to the potential of monosaccharide therapy for several other CDG.

A Bifunctional UDP-Sugar 4-Epimerase Supports Biosynthesis of Multiple Cell Surface Polysaccharides in Sinorhizobium meliloti.

Sinorhizobium meliloti produces multiple extracellular glycans, including among others, lipopolysaccharides (LPS), and the exopolysaccharides (EPS) succinoglycan (SG) and galactoglucan (GG). These polysaccharides serve cell protective roles. Furthermore, SG and GG promote the interaction of S. meliloti with its host Medicago sativa in root nodule symbiosis. ExoB has been suggested to be the sole enzyme catalyzing synthesis of UDP-galactose in S. meliloti (A. M. Buendia, B. Enenkel, R. Köplin, K. Niehaus, et al. Mol Microbiol 5:1519-1530, 1991, https://doi.org/10.1111/j.1365-2958.1991.tb00799.x). Accordingly, exoB mutants were previously found to be affected in the synthesis of the galactose-containing glycans LPS, SG, and GG and consequently, in symbiosis. Here, we report that the S. meliloti Rm2011 uxs1-uxe-apsS-apsH1-apsE-apsH2 (SMb20458-63) gene cluster directs biosynthesis of an arabinose-containing polysaccharide (APS), which contributes to biofilm formation, and is solely or mainly composed of arabinose. Uxe has previously been identified as UDP-xylose 4-epimerase. Collectively, our data from mutational and overexpression analyses of the APS biosynthesis genes and in vitro enzymatic assays indicate that Uxe functions as UDP-xylose 4- and UDP-glucose 4-epimerase catalyzing UDP-xylose/UDP-arabinose and UDP-glucose/UDP-galactose interconversions, respectively. Overexpression of uxe suppressed the phenotypes of an exoB mutant, evidencing that Uxe can functionally replace ExoB. We suggest that under conditions stimulating expression of the APS biosynthesis operon, Uxe contributes to the synthesis of multiple glycans and thereby to cell protection, biofilm formation, and symbiosis. Furthermore, we show that the C2H2 zinc finger transcriptional regulator MucR counteracts the previously reported CuxR-c-di-GMP-mediated activation of the APS biosynthesis operon. This integrates the c-di-GMP-dependent control of APS production into the opposing regulation of EPS biosynthesis and swimming motility in S. melilotiIMPORTANCE Bacterial extracellular polysaccharides serve important cell protective, structural, and signaling roles. They have particularly attracted attention as adhesives and matrix components promoting biofilm formation, which significantly contributes to resistance against antibiotics. In the root nodule symbiosis between rhizobia and leguminous plants, extracellular polysaccharides have a signaling function. UDP-sugar 4-epimerases are important enzymes in the synthesis of the activated sugar substrates, which are frequently shared between multiple polysaccharide biosynthesis pathways. Thus, these enzymes are potential targets to interfere with these pathways. Our finding of a bifunctional UDP-sugar 4-epimerase in Sinorhizobium meliloti generally advances the knowledge of substrate promiscuity of such enzymes and specifically of the biosynthesis of extracellular polysaccharides involved in biofilm formation and symbiosis in this alphaproteobacterium.

SLC35A2-CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals.

Pathogenic de novo variants in the X-linked gene SLC35A2 encoding the major Golgi-localized UDP-galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2-congenital disorders of glycosylation (CDG; formerly CDG-IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin N-glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2-CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2-dependent UDP-galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wild-type to mutant alleles in fibroblasts from affected individuals.

Repetitive Batch Mode Facilitates Enzymatic Synthesis of the Nucleotide Sugars UDP-Gal, UDP-GlcNAc, and UDP-GalNAc on a Multi-Gram Scale.

The availability of nucleotide sugars is considered as bottleneck for Leloir-glycosyltransferases mediated glycan synthesis. A breakthrough for the synthesis of nucleotide sugars is the development of salvage pathway like enzyme cascades with high product yields from affordable monosaccharide substrates. In this regard, the authors aim at high enzyme productivities of these cascades by a repetitive batch approach. The authors report here for the first time that the exceptional high enzyme cascade stability facilitates the synthesis of Uridine-5'-diphospho-α-d-galactose (UDP-Gal), Uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), and Uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in a multi-gram scale by repetitive batch mode. The authors obtained 12.8 g UDP-Gal through a high mass based total turnover number (TTNmass ) of 494 [gproduct /genzyme ] and space-time-yield (STY) of 10.7 [g/L*h]. Synthesis of UDP-GlcNAc in repetitive batch mode gave 11.9 g product with a TTNmass of 522 [gproduct /genzyme ] and a STY of 9.9 [g/L*h]. Furthermore, the scale-up to a 200 mL scale using a pressure operated concentrator was demonstrated for a UDP-GalNAc producing enzyme cascade resulting in an exceptional high STY of 19.4 [g/L*h] and 23.3 g product. In conclusion, the authors demonstrate that repetitive batch mode is a versatile strategy for the multi-gram scale synthesis of nucleotide sugars by stable enzyme cascades.

Profiling of intracellular metabolites produced from galactose and its potential for galactosemia research.

BACKGROUND: Clinical outcome of patients with a classical presentation of galactosemia (classical patients) varies substantially, even between patients with the same genotype. With current biomarkers, it is not possible to predict clinical outcome early in life. The aim of this study was to develop a method to provide more insight into galactose metabolism, which allows quantitative assessment of residual galactose metabolism in galactosemia patients. We therefore developed a method for galactose metabolite profiling (GMP) in fibroblasts using [U-13C]-labeled galactose. METHODS: GMP analysis was performed in fibroblasts of three classical patients, three variant patients and three healthy controls. The following metabolites were analyzed: [U13C]-galactose, [U13C]-galactose-1-phosphate (Gal-1-P) and [13C6]- uridine diphosphate(UDP)-galactose. The ratio of [U13C]-Gal-1-P/ [13C6]-UDP-galactose was defined as the galactose index (GI). RESULTS: All patient cell lines could be distinguished from the control cell lines and there was a clear difference between variant and classical patients. Variant patients had lower levels of [U13C]-galactose and [U13C]-Gal-1-P than classical patients (though substantially higher than healthy controls) and higher levels of [13C6]-UDP-galactose than classical patients (though substantially lower than healthy controls) resulting in a different GI in all groups. CONCLUSIONS: GMP in fibroblasts is a sensitive method to determine residual galactose metabolism capacity, which can discriminate between patients with a classical presentation of galactosemia, patients with a variant presentation and healthy controls. GMP may be a useful method for early prognostication after further validation in a larger cohort of patients representing the full phenotypic spectrum of galactosemia.

Steric Control of the Rate-Limiting Step of UDP-Galactopyranose Mutase.

Galactose is an abundant monosaccharide found exclusively in mammals as galactopyranose (Gal p), the six-membered ring form of this sugar. In contrast, galactose appears in many pathogenic microorganisms as the five-membered ring form, galactofuranose (Gal f). Gal f biosynthesis begins with the conversion of UDP-Gal p to UDP-Gal f catalyzed by the flavoenzyme UDP-galactopyranose mutase (UGM). Because UGM is essential for the survival and proliferation of several pathogens, there is interest in understanding the catalytic mechanism to aid inhibitor development. Herein, we have used kinetic measurements and molecular dynamics simulations to explore the features of UGM that control the rate-limiting step (RLS). We show that UGM from the pathogenic fungus Aspergillus fumigatus also catalyzes the isomerization of UDP-arabinopyranose (UDP-Ara p), which differs from UDP-Gal p by lacking a -CH2-OH substituent at the C5 position of the hexose ring. Unexpectedly, the RLS changed from a chemical step for the natural substrate to product release with UDP-Ara p. This result implicated residues that contact the -CH2-OH of UDP-Gal p in controlling the mechanistic path. The mutation of one of these residues, Trp315, to Ala changed the RLS of the natural substrate to product release, similar to the wild-type enzyme with UDP-Ara p. Molecular dynamics simulations suggest that steric complementarity in the Michaelis complex is responsible for this distinct behavior. These results provide new insight into the UGM mechanism and, more generally, how steric factors in the enzyme active site control the free energy barriers along the reaction path.

UDP-Glucose 4-Epimerase and ß-1,4-Galactosyltransferase from the Oyster Magallana gigas as Valuable Biocatalysts for the Production of Galactosylated Products.

Uridine diphosphate galactose (UDP-galactose) is a valuable building block in the enzymatic synthesis of galactose-containing glycoconjugates. UDP-glucose 4-epimerase (UGE) is an enzyme which catalyzes the reversible conversion of abundantly available UDP-glucose to UDP-galactose. Herein, we described the cloning, expression, purification, and biochemical characterization of an unstudied UGE from the oyster Magallana gigas (MgUGE). Activity tests of recombinantly expressed MgUGE, using HPLC (high-performance liquid chromatography), mass spectrometry, and photometric assays, showed an optimal temperature of 16 °C, and reasonable thermal stability up to 37 °C. No metal ions were required for enzymatic activity. The simple nickel-affinity-purification procedure makes MgUGE a valuable biocatalyst for the synthesis of UDP-galactose from UDP-glucose. The biosynthetic potential of MgUGE was further exemplified in a coupled enzymatic reaction with an oyster-derived ß-1,4-galactosyltransferase (MgGalT7), allowing the galactosylation of the model substrate para-nitrophenol xylose (pNP-xylose) using UDP-glucose as the starting material.

Solid-Phase Enzymatic Remodeling Produces High Yields of Single Glycoform Antibodies.

Antibodies are synthesized in mammalian cell culture as heterogeneous mixtures of glycoforms. Production of single glycoforms remains a challenge despite their value as therapeutics. The authors report a method of sequential enzymatic-based changes to antibodies while immobilized on an affinity column. Various antibodies (monoclonal and polyclonal) are isolated on Protein A or G columns and their glycans modified by sequential addition of enzymes for a desired transformation. Galactosylated antibodies (>90% yield) are produced by a one stage reaction process with sialidase to remove any sialic acid residues and addition of galactose with galactosyltransferase and UDP-Gal. Sialylated antibodies (>90%) are produced by a 2 stage conversion involving α(2,3) sialidase and galactosyltransferase followed by treatment with α(2,6) sialyltransferase in the presence of CMP-NANA. By this method, >90% of a disialylated human-llama antibody (EG2-hFc) and equimolar quantities of monosialylated and disialylated forms of human antibodies (αIL8-hFc and human polyclonal) are produced. Such high levels of sialylation are very difficult to obtain by typical cell culture methods. This method of transformation while the antibody is held on a solid-phase column is superior to previous methods because it allows a series of enzymatic steps without the need for intermediate purification. This is an efficient and rapid method to generate therapeutic antibodies with predefined glycosylation profiles. This should also assist in investigating the structure-function relationship of antibody glycans to find the desired glycosylation profile for high functional activity. With further optimization the method can be used to modify antibodies in large-scale manufacturing.