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.
SUMMARY
Rhamnogalacturonan‐II (RG‐II) is structurally the most complex glycan in higher plants, containing 13 different sugars and 21 distinct glycosidic linkages. Two monomeric RG‐II molecules can ...form an RG‐II‐borate diester dimer through the two apiosyl (Api) residues of side chain A to regulate cross‐linking of pectin in the cell wall. But the relationship of Api biosynthesis and RG‐II dimer is still unclear. In this study we investigated the two homologous UDP‐D‐apiose/UDP‐D‐xylose synthases (AXSs) in Arabidopsis thaliana that synthesize UDP‐D‐apiose (UDP‐Api). Both AXSs are ubiquitously expressed, while AXS2 has higher overall expression than AXS1 in the tissues analyzed. The homozygous axs double mutant is lethal, while heterozygous axs1/+ axs2 and axs1 axs2/+ mutants display intermediate phenotypes. The axs1/+ axs2 mutant plants are unable to set seed and die. By contrast, the axs1 axs2/+ mutant plants exhibit loss of shoot and root apical dominance. UDP‐Api content in axs1 axs2/+ mutants is decreased by 83%. The cell wall of axs1 axs2/+ mutant plants is thicker and contains less RG‐II‐borate complex than wild‐type Col‐0 plants. Taken together, these results provide direct evidence of the importance of AXSs for UDP‐Api and RG‐II‐borate complex formation in plant growth and development.
Significance Statement
This paper investigates the function of AXS in Arabidopsis. We emphasize the crucial role of AXS in plant development and reproduction by controlling UDP‐Api content to regulate RG‐II‐borate complex levels.
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.
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.
A novel human nucleotide sugar transporter (NST) which transports both UDP-glucuronic acid (UDP-GlcA) and UDP-
N-acetylgalactosamine (UDP-GalNAc) has been identified, cloned and characterized. The ...strategy for the identification of the novel NST involved a search of the expressed sequence tags database for genes related to the human UDP-galactose transporter-related isozyme 1, followed by heterologous expression of a candidate gene (hUGTrel7) in
Saccharomyces cerevisiae and biochemical analyses. Significantly more UDP-GlcA and UDP-GalNAc were translocated from the reaction medium into the lumen of microsomes prepared from the hUGTrel7-expressing yeast cells than into the control microsomes from cells not expressing hUGTrel7. The possibility that this transporter participates in glucuronidation and/or chondroitin sulfate biosynthesis is discussed.
UDP‐d‐glucuronic acid and UDP‐d‐xylose are required for the biosynthesis of glycosaminoglycan in mammals and of cell wall polysaccharides in plants. Given the importance of these glycans to some ...organisms, the development of a system for production of UDP‐d‐glucuronic acid and UDP‐d‐xylose from a common precursor could prove useful for a number of applications. The budding yeast Saccharomyces cerevisiae lacks an endogenous ability to synthesize or consume UDP‐d‐glucuronic acid and UDP‐d‐xylose. However, yeast have a large cytoplasmic pool of UDP‐d‐glucose that could be used to synthesize cell wall β‐glucan, as a precursor of UDP‐d‐glucuronic acid and UDP‐d‐xylose. Thus, if a mechanism for converting the precursors into the end‐products can be identified, yeast may be harnessed as a system for production of glycans. Here we report a novel S. cerevisiae strain that coexpresses the Arabidopsis thaliana genes UGD1 and UXS3, which encode a UDP‐glucose dehydrogenase (AtUGD1) and a UDP‐glucuronic acid decarboxylase (AtUXS3), respectively, which are required for the conversion of UDP‐d‐glucose to UDP‐d‐xylose in plants. The recombinant yeast strain was capable of converting UDP‐d‐glucose to UDP‐d‐glucuronic acid, and UDP‐d‐glucuronic acid to UDP‐d‐xylose, in the cytoplasm, demonstrating the usefulness of this yeast system for the synthesis of glycans. Furthermore, we observed that overexpression of AtUGD1 caused a reduction in the UDP‐d‐glucose pool, whereas coexpression of AtUXS3 and AtUGD1 did not result in reduction of the UDP‐d‐glucose pool. Enzymatic analysis of the purified hexamer His‐AtUGD1 revealed that AtUGD1 activity is strongly inhibited by UDP‐d‐xylose, suggesting that AtUGD1 maintains intracellular levels of UDP‐d‐glucose in cooperation with AtUXS3 via the inhibition of AtUGD1 by UDP‐d‐xylose.
Leishmania, a protozoan parasite that causes leishmaniasis, affects 1‐2 million people every year worldwide. Leishmaniasis is a vector born disease and characterized by a diverse group of clinical ...syndromes. Current treatment is limited because of drug resistance, high cost, poor safety, and low efficacy. The urgent need for potent agents against Leishmania has led to significant advances in the development of novel antileishmanial drugs. β‐galactofuranose (β‐Galf) is an important component of Leishmanial cell surface matrix and plays a critical role in the pathogenesis of parasite. UDP‐galactopyranose mutase (UGM) converts UDP‐galactopyranose (UDP‐Galp) to UDP‐galactofuranose (UDP‐Galf) which acts as the precursor for β‐Galf synthesis. Due to its absence in human, this enzyme is selected as the potential target in search of new antileishmanial drugs. Three dimensional protein structure model of Leishmania major UGM (LmUGM) has been homology modeled using Trypanosoma cruzi UGM (TcUGM) as a template. The stereochemistry was validated further. We selected already reported active compounds from PubChem database to target the LmUGM. Three compounds (6064500, 44570814, and 6158954) among the top hit occupied the UDP binding site of UGM suggested to work as a possible inhibitor for it. In vitro antileishmanial activity assay was performed with the top ranked inhibitor, 6064500. The 6064500 molecule has inhibited the growth of Leishmania donovani promastigotes significantly. Further, at similar concentrations it has exhibited significantly lesser toxicity than standard drug miltefosine hydrate in mammalian cells.
In this study, we have selected UDP‐galactopyranose mutase (UGM), an enzyme involved in the synthesis of β‐Galactofuranose (β‐Galf), a major component of leishmanial membrane. Due to its absence in human, this enzyme has been targeted to search new antileishmanial drugs. Three dimensional model of Leishmania major UGM (LmUGM) has been generated using Trypanosoma cruzi UGM (TcUGM) as template and screened for reported active compounds from PubChem database to target it. Inhibitor having pubchem id: 6064500 has been proven as the best screened ligand of Lm‐UGM which was further validated by in vitro antileishmanial activity assay. It has exhibited comparatively and significantly lesser toxicity than miltefosine hydrate (known antileishmanial drug) in mammalian cells when treated with the same concentrations. This is the first study targeting Lm‐UGM for the screening of the proposed potential inhibitors to treat leishmaniasis in the best of our knowledge.
•N-glycosylation deficient mutants were more susceptible against Pst DC3000 than wild type plants.•COS induces resistance to Pst DC3000 in N-glycosylation deficient mutants.•COS regulates ...N-glycosylation via a non-canonical pattern compared with Pst DC3000.
Roles of protein N-glycosylation in chitosan oligosaccharide (COS) induced resistance were investigated in the present study. Results demonstrated that N-glycosylation deficient Arabidopsis mutants (stt3a and ManI) were more susceptible against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) than wild type (WT) plants. Surprisingly, in stt3a and ManI, COS-induced resistance to Pst DC3000 was mostly intact, and the up-regulation effect on SA- and JA-mediated signalling pathways also similar like WT. Nucleotide sugars accumulation and N-glycosylation related genes expression were differently regulated after COS treatment. Global glycomics analysis quantified 157 N-glycan isomers, and 56.7, 50.3 and 47.1 % of them were significantly changed in COS, mock + Pst, and COS + Pst treated plants, respectively. Moreover, COS pretreatment could reverse the effect of Pst DC3000 on many N-glycans, suggesting that COS regulates protein N-glycosylation via a non-canonical pattern compared with plant defense, which may contribute to its obvious disease control effect when N-glycosylation impairment occurs.
UDP glucose pyrophosphorylase (UDPGP) family genes have been reported to play essential roles in cell death or individual survival. However, a systematic analysis on UDPGP gene family has not been ...performed yet. In this study, a total of 454 UDPGP proteins from 76 different species were analyzed. The analyses of the phylogenetic tree and orthogroups divided UDPGPs into three clades, including UDP-
-acetylglucosamine pyrophosphorylase (UAP), UDP-glucose pyrophosphorylase (UGP, containing UGP-A and UGP-B), and UDP-sugar pyrophosphorylase (USP). The evolutionary history of the UDPGPs indicated that the members of UAP, USP, and UGP-B were relatively conserved while varied in UGP-A. Homologous sequences of UGP-B and USP were found only in plants. The expression profile of UDPGP genes in
was mainly motivated under jasmonic acid (JA), abscisic acid (ABA), cadmium, and cold treatments, indicating that UDPGPs may play an important role in plant development and environment endurance. The key amino acids regulating the activity of UDPGPs were analyzed, and almost all of them were located in the NB-loop, SB-loop, or conserved motifs. Analysis of the natural variants of UDPGPs in rice revealed that only a few missense mutants existed in coding sequences (CDSs), and most of the resulting variations were located in the non-motif sites, indicating the conserved structure and function of UDPGPs in the evolution. Furthermore, alternative splicing may play a key role in regulating the activity of UDPGPs. The spatial structure prediction, enzymatic analysis, and transgenic verification of UAP isoforms illustrated that the loss of N- and C-terminal sequences did not affect the overall 3D structures, but the N- and C-terminal sequences are important for UAP genes to maintain their enzymatic activity. These results revealed a conserved UDPGP gene family and provided valuable information for further deep functional investigation of the UDPGP gene family in plants.
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