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.

A novel polygalacturonic acid bioflocculant REA-11 produced by Corynebacterium glutamicum: a proposed biosynthetic pathway and experimental confirmation.

Corynebacterium glutamicum CCTCC M201005 produces a novel polygalacturonic acid bioflocculant, REA-11, consisting of galacturonic acid as the main structural unit. A biosynthetic pathway of REA-11 in C. glutamicum CCTCC M201005 was proposed. Evidence for the biosynthetic pathway was provided by: (1) analyzing the response upon addition of UDP-glucose to the culture medium; (2) detecting the presence of several key intermediates in the pathway; and (3) correlating the activities of several key enzymes involved in the pathway with the yields of polygalacturonic acid. The production of polygalacturonic acid was improved by 24%, while the activities of UDP-galactose epimerase and UDP-galactose dehydrogenase were improved by 200% and 50%, respectively, upon addition of 100 microM UDP-glucose. In addition, the key intermediates in the proposed biosynthetic pathway, such as UDP-glucose, UDP-galactose, and UDP-glucuronic acid, were detected in cell-free extracts. Furthermore, the activities of UDP-glucose pyrophosphorylase (R2=0.97), UDP-galactose epimerase (R2=0.75) and UDP-galactose dehydrogenase (R2=0.89) were well correlated with the yields of polygalacturonic acid when different sugars were used as sole carbon sources. Therefore, the biosynthetic pathway of REA-11 in C. glutamicum CCTCC M201005 starts from phosphate-1-glucose, which was then converted to UDP-glucose by UDP-pyrophosphorylase. Predominantly, the UDP-glucose was converted to UDP-galactose by UDP-galactose epimerase; the latter was further converted to UDP-galacturonic acid by UDP-galactose dehydrogenase, which was presumably polymerized to polygalacturonic acid bioflocculant REA-11 by an unknown glucosyltransferase and a polymerase.

Solubilization of rhamnogalacturonan I galactosyltransfrases from membranes of a flax cell suspension.

Galactosyltransferases (GalTs), capable of transferring a galactosyl residue from UDP-galactose (UDP-Gal) to polysaccharide acceptor, were solubilized from flax (Linum usitatissimum L.) membranes using 0.5% CHAPS. The observed requirement for a rhamnogalacturonan I (RG-I) exogenous substrate to stimulate the solubilized GalT activity provided the first evidence for the presence of RG-I GalT activities in flax cells. An assay to measure specifically the products of this RG-I GalT activity was designed, based on size-exclusion chromatography. Labelled products were characterized as an RG-I polymer by using purified RG-I hydrolase or lyase. At pH 8 and in the presence of 5 mM CaCl2, beta-D-galactosyl residues were specifically transferred onto RG-I branches of short beta-(1 --> 4)-D-galactan side chains. These side chains were liable to hydrolysis by beta-galactosidase and endo-beta-(1 --> 4)-D-galactanase. The RG-I GalT had a temperature optimum of 30 degrees C. an apparent Km for UDP-Gal and exogenous RG-I substrate of 460 +/- 40 microM and 1.1 +/- 0.1 mg ml(-1) respectively, and a Vmax of 3.0 +/- 0.5 pkat mg(-1) protein.

Spontaneous galactosylation of agalactoglycoproteins in colostrum.

We have found that spontaneous galactosylation of GlcNAc residues occurs in bovine colostrum, but not in dialyzed colostrum, without adding UDP-Gal as a donor substrate. UDP-Gal was shown to be present in bovine colostrum at a level ranging from 200 to 600 microM. When a tracer UDP-[(14)C]Gal was added to the dialyzed colostrum together with a Gal beta1,4-specific beta-galactosidase, remarkable incorporation of radioactivity into 24-28 kDa and 33 kDa RCA1-positive glycoproteins was demonstrated by SDS-PAGE/autoradiography. Some 100-140 kDa agalactoglycoproteins of a CHO mutant cell line were also galactosylated on a blotted membrane by the incubation in the colostrum.

Effect of organic cosolvents on the stability and activity of the beta-1,4-galactosyltransferase from bovine colostrum.

The influence of various organic cosolvents on the stability and activity of the beta-1,4-galactosyltransferase from bovine colostrum (GalT) and of its ancillary enzyme UDP-galactose-4'-epimerase has been investigated using the glucosylated alkaloid colchicoside (1) as a model substrate. It has been found that some cosolvents, such as Me2SO and MeOH, can be used up to 20% v/v without any influence on the performance of these enzymes, while others, such as tetrahydrofuran, rapidly inactivated GalT at concentrations as low as 5% v/v. These results have been exploited for the galactosylation of the poorly water soluble coumarinic glucoside fraxin (2).

Sugar nucleotide concentrations in red blood cells of patients on protein- and lactose-limited diets: effect of galactose supplementation.

Uridine diphosphate (UDP) galactose, a pivotal compound in the metabolism of galactose, is the obligate donor of galactose in the formation of complex glycoconjugates. The cellular UDPgalactose concentration has been thought to be maintained by the interconversion of UDPglucose and UDPgalactose by UDPgalactose-4-epimerase. However, recent findings of lower average red blood cell (RBC) UDPgalactose concentrations in galactose-1-phosphate uridyltransferase-deficient patients suggest that other factors play a role in determining its concentration. To test the hypothesis that the amount of galactose traversing the Leloir pathway contributes to the cellular UDPgalactose pool, we determined RBC UDPgalactose in patients with maple syrup urine disease (MSUD), phenylketonuria (PKU), and other metabolic diseases who were treated with a low-protein, and consequently, low-lactose diet. Six patients with MSUD were also supplemented with 19 g galactose/d and their UDPhexose concentrations were measured at intervals. We show that young patients with MSUD or PKU have decreased average RBC UDPgalactose concentrations when compared with similarly aged healthy subjects. Galactose supplementation of MSUD patients significantly increased their UDPgalactose concentrations in both RBCs and white blood cells (WBCs) from 29.5 +/- 1.5 to 42.3 +/- 5.8 nmol/g hemoglobin and from 69.0 +/- 7.5 to 193.0 +/- 49.0 nmol/g protein, respectively. Discontinuation of supplementation was associated with a return to basal values in RBCs and a reattainment of the pretreatment ratio of UDPglucose to UDPgalactose in WBCs. These observations demonstrate that dietary galactose is a factor in establishing the steady state concentrations of the uridine sugar nucleotides and imply that galactose metabolism modulates the achievement of an epimerase-mediated equilibrium.

Use of site-directed mutagenesis to identify the galactosyltransferase binding sites for UDP-galactose.

Site-directed mutagenesis was utilized to identify binding sites for UDP-galactose in galactosyltransferase (EC 2.4.1.22). Mutant cDNAs were generated by a procedure based on PCR, and the mutated enzymes were expressed in E.coli cells. The mutant enzymes were purified by Ni-NTA Sephadex, and the degree of purification was judged by SDS-PAGE. Purified mutant GTs, F305L, P306V, N307S, N308S, showed dramatic decreases in activities in comparison with the activity of the wild-type GT. Enzyme kinetic analysis revealed that the Km values of F305L, P306V, N307S and N308S for UDP-galactose were, respectively, 9-, 11-, 50- and 20-fold higher than the Km of wild-type GT, but the Km values for manganese were not significantly different from that of the wild-type GT. The quartet mutant F305L/P306V/N307S/N308S showed no activity. From the results of this study it is concluded that amino acids, Phe-305, Pro-306, Asn-307 and Asn-308, in GT are most probably involved in GT catalysis or are located close to the UDP-galactose binding region but are not involved in the binding of manganese.

Identification of a region of UDP-galactose:N-acetylglucosamine beta 4-galactosyltransferase involved in UDP-galactose binding by differential labeling.

The location of regions in the primary structure of UDP-galactose:N-acetylglucosamine beta 4-galactosyl-transferase (GT) that are involved in binding UDP-galactose has been investigated by differential chemical modification with two different reagents in the presence and absence of UDP-galactose. Treatment with periodate-cleaved UDP and NaCNBH3 resulted in a loss of 80% of GT activity, which was largely prevented by UDP-galactose. Stoichiometry of labeling and peptide maps of the modified enzyme samples indicated partial labeling at many sites. A major site of reaction in the absence of UDP-galactose that was essentially unmodified in its presence was found to correspond to Lys341 in the cDNA sequence of GT. As a second approach, the reactivities of the amino groups of GT were compared in the presence and absence of saturating levels of UDP-galactose by trace acetylation with [3H]acetic anhydride. UDP-galactose binding was found to perturb the reactivities of a number of lysines in the C-terminal region of GT, the most pronounced effect being a reduction in the reactivity of Lys351. The two procedures thus identified a region between residues 341 and 351 as being associated with UDP-galactose binding. This region overlaps a small section in the sequence of GT that was previously noted to be similar to part of bovine alpha-1,3-galactosyltransferase (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). Sequence comparisons indicate that extended regions at the C terminus of each enzyme encompassing this area may represent homologous UDP-galactose-binding domains.

Affinity labeling of bovine colostrum galactosyltransferase with a uridine 5'-diphosphate derivative.

The dialdehyde produced by the periodate cleavage of the ribose moiety of uridine 5'-diphosphate (UDP) has been used as an affinity label for the UDP-galactose/UDP binding site of galactosyltransferase from bovine colostrum. This derivative causes progressive inactivation of galactosyltransferase at a rate dependent on its concentration, and under certain conditions is a competitive inhibitor with respect to UDP-galactose. The substrate UDP-galactose protects the enzyme from inactivation. The inactivation is also dependent on Mn2+ concentration in a range that implies that the binding of Mn2+ at site I is a prerequisite for the binding of the UDP derivative. The inactivation can be progressively reversed by nitrogenous bases, or stabilized by KBH4 reduction, which is consistent with the hypothesis that a Schiff base has formed with a lysine residue. Galactosyltransferase was inactivated with a [3H]UDP derivative and the predominant labeled peptide, from thermolysin digestion, isolated and characterized as: Ser-Gly-Lys-UDP.