Summary
Glycosylated metabolites generated by UDP‐dependent glycosyltransferases (UGTs) play critical roles in plant interactions with the environment as well as human and animal nutrition. The ...evolution of plant UGTs has previously been explored, but with a limited taxon sampling. In this study, 65 fully sequenced plant genomes were analyzed, and stringent criteria for selection of candidate UGTs were applied to ensure a more comprehensive taxon sampling and reliable sequence inclusion. In addition to revealing the overall evolutionary landscape of plant UGTs, the phylogenomic analysis also resolved the phylogenetic association of UGTs from free‐sporing plants and gymnosperms, and identified an additional UGT group (group R) in seed plants. Furthermore, lineage‐specific expansions and contractions of UGT groups were detected in angiosperms, with the total number of UGTs per genome remaining constant generally. The loss of group Q UGTs in Poales and Brassicales, rather than functional convergence in the group Q containing species, was supported by a gene tree of group Q UGTs sampled from many species, and further corroborated by the absence of group Q homologs on the syntenic chromosomal regions in Arabidopsis thaliana (Brassicales). Branch‐site analyses of the group Q UGT gene tree allowed for identification of branches and amino acid sites that experienced episodic positive selection. The positively selected sites are located on the surface of a representative group Q UGT (PgUGT95B2), away from the active site, suggesting their role in protein folding/stability or protein–protein interactions.
Significance Statement
This phylogenomic analysis identified additional phylogenetic groups of plant UGTs, resolved the phylogenetic association of UGTs from free‐sporing plants and gymnosperms, revealed lineage‐specific expansions and contractions of UGT groups in angiosperms, and identified positively selected branches and amino acid sites in group Q UGTs. Understanding the evolution of UGTs functioning in plant metabolism provides insights into plant–environment interactions, human and animal nutrition, and pharmaceutical development, as well as the evolution of enzymes, pathways and genomes.
Summary
Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously ...identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side‐effects. Here, we report that UDP‐glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP‐glucuronic acid to glycyrrhetinic acid 3‐O‐monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin‐deficient (83‐555) strain of G. uralensis. The natural variant showed loss of specificity for UDP‐glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83‐555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP‐glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP‐glucuronic acid. The functional arginine residue and resultant specificity for UDP‐glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP‐sugar selectivity for the rational engineering of sweet triterpenoid saponins.
Significance Statement
Glycyrrhizin, a licorice‐derived sweet triterpenoid diglycoside, is one of the most important phytoconstituents in the pharmaceutical and food industries. Here, we identified UGT73P12 as the enzyme catalyzing the sugar–sugar linkage to produce glycyrrhizin from its less glycosylated precursor in the presence of UDP‐glucuronic acid, and found that Arg32 of UGT73P12 is essential for the high specificity for UDP‐glucuronic acid, which was probably acquired during evolution to enable the functional specialization of UGT73P12 for glycyrrhizin biosynthesis.
Synthetic ways towards uridine 5′‐diphosphate (UDP)‐xylose are scarce and not well established, although this compound plays an important role in the glycobiology of various organisms and cell types. ...We show here how UDP‐glucose 6‐dehydrogenase (hUGDH) and UDP‐xylose synthase 1 (hUXS) from Homo sapiens can be used for the efficient production of pure UDP‐α‐xylose from UDP‐glucose. In a mimic of the natural biosynthetic route, UDP‐glucose is converted to UDP‐glucuronic acid by hUGDH, followed by subsequent formation of UDP‐xylose by hUXS. The nicotinamide adenine dinucleotide (NAD+) required in the hUGDH reaction is continuously regenerated in a three‐step chemo‐enzymatic cascade. In the first step, reduced NAD+ (NADH) is recycled by xylose reductase from Candida tenuis via reduction of 9,10‐phenanthrenequinone (PQ). Radical chemical re‐oxidation of this mediator in the second step reduces molecular oxygen to hydrogen peroxide (H2O2) that is cleaved by bovine liver catalase in the last step. A comprehensive analysis of the coupled chemo‐enzymatic reactions revealed pronounced inhibition of hUGDH by NADH and UDP‐xylose as well as an adequate oxygen supply for PQ re‐oxidation as major bottlenecks of effective performance of the overall multi‐step reaction system. Net oxidation of UDP‐glucose to UDP‐xylose by hydrogen peroxide (H2O2) could thus be achieved when using an in situ oxygen supply through periodic external feed of H2O2 during the reaction. Engineering of the interrelated reaction parameters finally enabled production of 19.5 mM (10.5 g L−1) UDP‐α‐xylose. After two‐step chromatographic purification the compound was obtained in high purity (>98%) and good overall yield (46%). The results provide a strong case for application of multi‐step redox cascades in the synthesis of nucleotide sugar products.
Numerous different nucleotide sugars are used as sugar donors for the biosynthesis of glycans by bacteria, humans, fungi, and plants. However, many of these nucleotide sugars are not available either ...in their native form or with the sugar portion labeled with a stable or radioactive isotope. Here we demonstrate the use of Escherichia coli metabolically engineered to contain genes that encode proteins that convert monosaccharides into their respective monosaccharide-1-phosphates and subsequently into the corresponding nucleotide sugars. In this system, which we designated “in-microbe”, reactions occur within 2 to 4h and can be used to generate nucleotide sugars in amounts ranging from 5 to 12.5μg/ml cell culture. We show that the E. coli can be engineered to produce the seldom observed nucleotide sugars UDP–2-acetamido-2-deoxy-glucuronic acid (UDP–GlcNAcA) and UDP–2-acetamido-2-deoxy-xylose (UDP–XylNAc). Using similar strategies, we also engineered E. coli to synthesize UDP–galacturonic acid (UDP–GalA) and UDP–galactose (UDP–Gal). 13C- and 15N-labeled NDP–sugars are formed using 13C glucose as the carbon source and with 15NNH4Cl as the nitrogen source.
The crystal structure of UDP‐N‐acetylglucosamine 4‐epimerase (UDP‐GlcNAc 4‐epimerase; WbpP; EC 5.1.3.7), from the archaeal methanogen Methanobrevibacter ruminantium strain M1, was determined to a ...resolution of 1.65 Å. The structure, with a single monomer in the crystallographic asymmetric unit, contained a conserved N‐terminal Rossmann‐fold for nucleotide binding and an active site positioned in the C‐terminus. UDP‐GlcNAc 4‐epimerase is a member of the short‐chain dehydrogenases/reductases superfamily, sharing sequence motifs and structural elements characteristic of this family of oxidoreductases and bacterial 4‐epimerases. The protein was co‐crystallized with coenzyme NADH and UDP‐N‐acetylmuramic acid, the latter an unintended inclusion and well known product of the bacterial enzyme MurB and a critical intermediate for bacterial cell wall synthesis. This is a non‐native UDP sugar amongst archaea and was most likely incorporated from the E. coli expression host during purification of the recombinant enzyme.
UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the ...action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and
(two isozymes) UDP-glucose pyrophosphorylases (UGPase),
UDP-sugar pyrophosphorylase (USPase) and
UDP-
-acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (
values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (
of 1.3 mM), β-L-Ara-1-P and α-D-Fuc-1-P (
of 3.4 mM), but not β-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (
of 1 mM) and, to some extent, D-Glc-1-P (
of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products.
Summary
Botrytis cinerea is a model plant‐pathogenic fungus that causes grey mould and rot diseases in a wide range of agriculturally important crops. A previous study has identified two enzymes and ...corresponding genes (bcdh, bcer) that are involved in the biochemical transformation of uridine diphosphate (UDP)‐glucose, the major fungal wall nucleotide sugar precursor, to UDP‐rhamnose. We report here that deletion of bcdh, the first biosynthetic gene in the metabolic pathway, or of bcer, the second gene in the pathway, abolishes the production of rhamnose‐containing glycans in these mutant strains. Deletion of bcdh or double deletion of both bcdh and bcer has no apparent effect on fungal development or pathogenicity. Interestingly, deletion of the bcer gene alone adversely affects fungal development, giving rise to altered hyphal growth and morphology, as well as reduced sporulation, sclerotia production and virulence. Treatments with wall stressors suggest the alteration of cell wall integrity. Analysis of nucleotide sugars reveals the accumulation of the UDP‐rhamnose pathway intermediate UDP‐4‐keto‐6‐deoxy‐glucose (UDP‐KDG) in hyphae of the Δbcer strain. UDP‐KDG could not be detected in hyphae of the wild‐type strain, indicating fast conversion to UDP‐rhamnose by the BcEr enzyme. The correlation between high UDP‐KDG and modified cell wall and developmental defects raises the possibility that high levels of UDP‐KDG result in deleterious effects on cell wall composition, and hence on virulence. This is the first report demonstrating that the accumulation of a minor nucleotide sugar intermediate has such a profound and adverse effect on a fungus. The ability to identify molecules that inhibit Er (also known as NRS/ER) enzymes or mimic UDP‐KDG may lead to the development of new antifungal drugs.
The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner ...side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid and (4) d-glutamic acid and dipeptide d-alanyl- d-alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.
Pentacyclic triterpenoids have wide applications in the pharmaceutical industry. The precise glucosylation at C‐3 OH of pentacyclic triterpenoids mediated by uridine 5'‐diphospho‐glucosyltransferase ...(UDP‐glucosyltransferase UGT) is an important way to produce valuable derivatives with various improved functions. However, most reported UGTs suffer from low regiospecificity toward the OH and COOH groups of pentacyclic triterpenoids, which significantly decreases the reaction efficiency. Here, two new UGTs (UGT73C33 and UGT73F24) were discovered in Glycyrrhiza uralensis. UGT73C33 showed high activity but poor regioselectivity toward the C‐3 OH and C‐30 COOH of pentacyclic triterpenoid, producing three glucosides. UGT73F24 showed rigid regioselectivity toward C‐3 OH of typical pentacyclic triterpenoids producing only C‐3 O‐glucosylated derivatives. In addition, UGT73C33 and UGT73F24 showed a broad substrate scope toward typical flavonoids with various sugar donors. Next, the substrate recognition mechanism of UGT73F24 toward glycyrrhetinic acid (GA) and UDP‐glucose was investigated. Two key residues, I23 and L84, were identified to determine activity, and site‐directed mutagenesis of UGT73F24‐I23G/L84N increased the activity by 4.1‐fold. Furthermore, three in vitro GA glycosylation systems with UDP‐recycling were constructed, and high yields of GA‐3‐O‐Glc (1.25 mM), GA‐30‐O‐Glc (0.61 mM), and GA‐di‐Glc (0.26 mM) were obtained. The de novo biosynthesis of GA‐3‐O‐glucose (26.31 mg/L) was also obtained in engineered yeast.
This work discovered two new UDP‐glucosyltransferases from Glycyrrhiza uralensis. UGT73C33 showed low regioselectivity toward C‐3 OH and C‐30 COOH of typical pentacyclic triterpenoids, but UGT73F24 showed rigid regioselectivity toward C‐3 OH of typical pentacyclic triterpenoids. Meanwhile, the critical residues responsible for determining activity of UGT73F24 were identified, which helped understand the molecular basis of UGT73F subfamily members. With UGT73F24 and its variant with improved activity, both in vitro and in vivo controllable glycosylation systems for pentacyclic triterpenoids were established.
Nucleotide sugar transporters (NST) mediate the transfer of nucleotide sugars from the cytosol into the lumen of the endoplasmatic reticulum and the Golgi apparatus. Because the NSTs show ...similarities with the plastidic phosphate translocators (pPTs), these proteins were grouped into the TPT/NST superfamily. In this study, a member of the NST-KT family, AtNST-KT1, was functionally characterized by expression of the corresponding cDNA in yeast cells and subsequent transport experiments. The histidine-tagged protein was purified by affinity chromatography and reconstituted into proteoliposomes. The substrate specificity of AtNST-KT1 was determined by measuring the import of radiolabelled nucleotide mono phosphates into liposomes preloaded with various unlabelled nucleotide sugars. This approach has the advantage that only one substrate has to be used in a radioactively labelled form while all the nucleotide sugars can be provided unlabelled. It turned out that AtNST-KT1 represents a monospecific NST transporting UMP in counterexchange with UDP-Gal but did not transport other nucleotide sugars. The
AtNST-KT1 gene is ubiquitously expressed in all tissues. AtNST-KT1 is localized to Golgi membranes. Thus, AtNST-KT1 is most probably involved in the synthesis of galactose-containing glyco-conjugates in plants.
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