Characterization of UDP-glucose pyrophosphorylases from different organisms Open Access

Nucleotide sugars play essential roles in carbohydrate metabolism, serving as glycosyl donors for the biosynthesis of polysaccharides, glycoproteins, and glycoconjugates. Among nucleotide sugars, UDP-sugars stand out—not only for the significance of the functions they are involved in the biosynthesis of glycans but also for their abundance (UDP-sugars make up as much as 55% of the total nucleotide pools) and the large number of reactions in which they are used as substrates [1,2]. For example, UDP-glucose (UDP-Glc) alone is involved in 270 reactions [3], acting as an essential precursor for the biosynthesis of glycans, e.g. sucrose, cellulose, hemicellulose, and glycogen, and glycan parts of glycoproteins, glycolipids, and proteoglycans in different organisms. Another role for UDP-Glc is to serve as a substrate for glycosylation of many secondary metabolites such as steroids, flavonoids, phenylpropanoids, betalains, terpenoids, and glucosinolates. In addition, UDP-Glc acts as a substrate for the glycosylation of numerous hormones (e.g. steroids, flavonoids, and phenylpropanoids) or glycosyl-hormone conjugates (e.g. auxin, cytokinin, and salicylic acid) [2].

UDP-glucose pyrophosphorylase (UGPase), also known as UTP-glucose-1-phosphate uridylyltransferase (EC 2.7.7.9), catalyzes a reversible production of UDP-Glc and pyrophosphate (PPi) from glucose-1-phosphate (Glc1P) and UTP. UGPase is a member of the NTP:sugar nucleotidyltransferases superfamily, a related group of proteins with a similar folding pattern and some conserved sequence elements [4]. The UGPases have essential activity for cell metabolism. The UGPases are not only related to the biosynthesis of glycans in microorganisms, plants, and animals but also linked to microorganism survival, plant programmed cell death, and even human cancers [5-7].

Despite the ubiquitous distribution of UGPase activity throughout all domains of life, prokaryotic UGPases are evolutionarily unrelated to their eukaryotic counterparts. The similarities between UGPases of prokaryotic origin and their eukaryotic counterparts are only ~8%, which is considered non-significant [4,8]. However, UGPases are very well conserved within the prokaryotic or eukaryotic kingdom. For instance, Escherichia coli UGPase (GalU) bears a 43.4% identity with UGPase (YngB) from the Bacillus subtilis, and the human UGPase (UGP2) and Arabidopsis thaliana UGPase share a 55.0% identity. Prokaryotic and eukaryotic UGPases differ significantly in protein size and tertiary and quaternary structure. The molecular mass of the prokaryotic UGPase monomer is approximately 30–35 kDa and crystallized as tetramers [4]. E. coli GalU (32.9 kDa) forms a tetramer with 222-point group symmetry; each monomer is dominated by an eight-stranded mixed β-sheet [9]. Molecular masses of eukaryotic UGPases are higher than those of prokaryotic UGPase. Molecular masses of plant UGPases protein are usually 50–55 kDa, depending on plant species [2,10,11]. The budding yeast Saccharomyces cerevisiae UGPase (Ugp1) (56.0 kDa) forms an octamer as the enzymatically active form of the protein [12]. Plants often contain UGPase isozymes. For example, there are two UGPase isozymes from A. thaliana, UGP1 and UGP2; the specific activity of UGP2 is higher than that of UGP1 [13].

The catalytic activity of UGPases from different organisms has been studied, such as prokaryotic UGPases from E. coli [14], B. subtilis [15], Xanthomonas citri [16], and Sulfurisphaera tokodaii [17] and eukaryotic UGPases from S. cerevisiae [18], A. thaliana [19], Hordeum vulgare [20], Solanum tuberosum [21,22], Leishmania major [23], and human [24]. However, it is not easy to compare the activity of UGPases from different organisms due to variations in assay methods. The activity of A. thaliana UGPase was determined by monitoring UDP-Glc produced by coupled enzymatic assays (UDP-Glc dehydrogenase was added) [19], and the activity of E. coli UGPase was determined by monitoring the amount of Glc1P with Mg²⁺-dependent phosphoglucomutase and glucose 6-phosphate dehydrogenase added [14]. In recent years, researchers have begun quantifying the Pi released from inorganic PPi using the colorimetric method to determine the activities of UGPases from B. subtilis, Brachypodium distachyon, Euglena gracilis, and humans [15,24-26]. However, these variations in assay methods have led to variations in measured activity value, even for the UGPase from the same species. For instance, the measured specific activity difference for the potato UGPase is greater than four times recorded by different research teams; the Michaelis constant K_m toward the same substrate UTP varies from 0.22 mM to 0.53 mM [27,28].

In this study, 11 UGPases from various organisms, including bacteria, fungi, plants, and animals, were expressed in E. coli. The recombinant UGPases were purified, and their specific activities were determined and compared. Four previously unstudied UGPases that are from Aspergillus niger, cassava, sweet potato, and maize were characterized. It has been discovered that MeUGP has the highest recorded specific activity to date.

The encoding sequences of UGPase from E. coli, S. cerevisiae, A. niger, A. thaliana, H. vulgare (barley), S. tuberosum (potato), Manihot esculenta (cassava), Ipomoea batatas (sweet potato), Zea mays (maize), Drosophila melanogaster (fruit fly), and Homo sapiens (human) were synthesized by GenScript Biotech Corporation (Nanjing, China) after codon optimization for heterologous expression in E. coli. The UniProt Entry of each UGPase can be found in Table 1. The sequences were digested with restriction endonucleases SalI and XhoI and ligated with plasmid pET28b(+) cut with the same restriction enzymes. For protein expression, the resulting recombinant plasmids were transformed into E. coli BL21 (DE3).

The fresh recombinant E. coli BL21 (DE3) cells were inoculated in 50-ml LB containing 30 mg/ml kanamycin in a 250-ml Erlenmeyer flask. The cells were incubated with shaking at 37°C until OD₆₀₀ reached 0.6–0.8. Protein expression was induced with a final concentration of 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 18°C for 16 h. After that, cells were harvested by centrifugation at 14000×g, 4°C for 15 min, and pellets were washed once with phosphate-buffered saline and resuspended in 5 ml of lysis buffer (50 mM Tris, 500 mM NaCl, pH7.0). The lysis buffer was added with RNase A to a final concentration of 100 µg/ml and protease inhibitor (Protease Inhibitor Cocktail I, 100× in DMSO, TargetMo). The cells were lysed by sonification with a microtip on ice. The cell lysate was centrifuged at 14000×g, 4°C for 15 min. The supernatant was collected and filtered through 0.22-µm filters (Merck Millipore). Clarified supernatant was loaded onto a 1-ml HisTra^™ HP column (Smart-Lifesciences, Changzhou, China) previously equilibrated with buffer A (50 mM Tris; 500 mM NaCl, pH 7.0). Then, the column was washed with 5 ml of buffer A, followed by buffer A containing 500 mM imidazole. The HisTrap flow-through was dialyzed in the buffer (50 mM Tris 150 mM NaCl, pH 7.0). Protein content was determined spectrophotometrically at 595 nm using the Modified Bradford Protein Assay Kit (Sango Biotech, Shanghai, China), with bovine serum albumin (BSA) as standard.

The activity of UGPase was determined using an assay based on quantification of the Pi released from inorganic PPi, the product of the pyrophosphorylase reaction according to the method of Wu et al. [15,25]. The reaction mixture contained 50 mM MOPS-KOH buffer, pH 7.0, 10 mM MgCl₂, 0.2 mg/ml BSA, 0.5 mM Glc1P, 1 mM UTP, 0.5 mU/ml of yeast inorganic pyrophosphatase, and a proper enzyme dilution. Assays were initiated by adding 0.5 mM Glc1P, incubated for 5 min at 37°C, and terminated by adding a Malachite Green Phosphate Detection Kit (Beyotime Biotechnology, Shanghai, China). The absorbance at 630 nm was measured with a Microplate Spectrophotometer (Agilent BioTek, Beijing, China) to determine the amount of the blue-colored phosphate–molybdenum complex proportional to the phosphate present. The amount of produced Pi was quantified with a Pi standard curve. One unit (U) of enzyme activity was defined as the amount of enzyme that produced 1 µmol of PPi per minute under the above reaction conditions.

The optimal temperature for UGPase activity was determined in different assay temperatures, ranging from 20℃ to 80℃ for 5 min. The optimal pH for UGPase activity was determined according to the method described by Decker et al. [32]. Prior to assays of the pH optimum, the buffer (12.5 mM acetate, 12.5 mM Hepes, 12.5 mM Mes, 12.5 mM Tricine, 10 mM MgCl₂, 0.2 mM Glc1P, 0.2 mM UTP, and an aliquot of purified UGPase) was prepared and adjusted to the required pH with 1 M NaOH or 1 M HCl and then was incubated for 5 min at 37°C. The assay procedures were as described in the ‘UGPase activity assay’ section. The thermostability of recombinant UGPase was determined by incubation with UGPases at 37°C, 50°C, 60°C, and 70°C. Purified enzymes of UGPase were diluted in 50 mM MOPS-KOH sodium acetate buffer (pH 7) and incubated at 37°C, 50°C, 60°C, and 70°C for various lengths of time. After heat treatment, the UGPases were tested as in the ‘UGPase activity assay’ section. The initial activity (unheated) was regarded as 100% activity. Residual activity was determined as a proportion of the initial activity.

kinetic constants were determined by measuring enzyme activity at different concentrations of one substrate while keeping a fixed and saturating amount of the other. for measuring the K_m value against the substrate glc1p, the reactions contained 0.2 mm utp and glc1p at a concentration of 2 mm, 1 mm, 0.5 mm, 0.2 mm, 0.1 mm, 0.05 mm, and 0.02 mm. the kinetic constants were calculated using the fitting software (graphpad prism). substrate specificity was determined by measuring enzyme activity toward nucleotide triphosphates and sugar phosphates. utp, atp, gtp, and ctp were used to investigate the specificity of ugpases toward nucleotide triphosphates. a quantity of 0.2 mm of utp, atp, gtp, or ctp was added to the mixtures containing 0.2 mm glc1p. glc1p, galactose 1-phosphate (gal1p), glucuronic acid 1-phosphate (glca1p), and n-acetyl-glucosamine 1-phosphate (glcnac1p) were used to investigate the specificity of ugpases toward sugar phosphates. a quantity of 0.2 mm of glc1p, gal1p, glca1p, or glcnac1p was added to the mixtures containing 0.2 mm utp. ppi generation indirectly indicates whether and to what extent ugpase binds to distinct substrates.

The protein sequences of various UGPases were downloaded from the UniProt database (https://www.uniprot.org/). Sequences were analyzed and aligned in the ClustalW server (http://www.genome.jp/tools/clustalw/). The phylogenetic tree was constructed using Molecular Evolutionary Genetic Analysis (MEGA 7.0) [33] with the Neighbor-Joining method, with a bootstrap of 1000.

The sources and nomenclatures of UGPase in this study are listed in Table 1. Eleven UGPases from different organisms were selected, including one UGPase from the bacteria E. coli (EcUGP); two UGPases from fungi S. cerevisiae (ScUGP) and A. niger (AnUPG); six UGPases from plants H. vulgare (barley) (HvUGP), A. thaliana (AtUGP), S. tuberosum (potato) (StUGP), M. esculenta (cassava) (MeUGP), I. batatas (sweet potato) (IbUGP), and Z. mays (maize) (ZmUGP); and two UGPases from animals D. melanogaster (fruit fly) (DmUGP) and H. sapiens (human) (HsUGP). Phylogenetic analysis shows that fungal UGPases and mammalian UGPases are more closely related to each other phylogenetically than to plant UGPases. There is no phylogenetic relationship between EcUGPase and UGPases from eukaryotic species (Figure 1), which is consistent with previous reports that prokaryotic UGPs are evolutionarily unrelated to their eukaryotic counterparts [4,8].

Among 11 UGPases, 7 UGPases (EcUGP, ScUGP, HvUGP, AtUGP, StUGP, DmUGP, and HsUGP) have been characterized previously (see the references in Table 1). These seven UGPases were chosen for two reasons: (1) The organisms that produce these seven UGPases cover kingdoms of bacteria, fungi, plants, and animals. E. coli, S. cerevisiae, A. thaliana, and D. melanogaster are model organisms; (2) UGPases from barley and potato have been the most extensively studied representatives of the plant UDP-sugar-producing family [20,32,34]. Four UGPases (AnUGP, MeUGP, IbUGP, and ZmUGP) have not yet been the subject of any research reports. Among them, one (AnUGP) from the fungus and three (MeUGP, IbUGP, and ZmUGP) from plants were chosen for two reasons: (1) Fungus A. niger is considered generally recognized as safe and is one of the most important microorganisms used in biotechnology to produce protein, food enzymes, organic acids, and secondary metabolites [35,36]. UGPase may become the engineering target in the modification of A. niger as chassis by metabolic engineering; (2) ADP-GPase is plastidic starch phosphorylase, which supplies ADP-glucose as the glucosyl donor to elongate alpha-1,4-glucosidic chains in plant starch synthesis. Interestingly, there is a high level of specific activity of plant UGPases, such as UGPase from barley and potato, as indicated by the data of the BRENDA database (https://www.brenda-enzymes.org/index.php). Therefore, special attention was given to the plant UGPases, especially plants with higher starch content (cassava, sweet potato, and maize).

Eleven UGPases from different organisms were expressed using E. coli BL21 (DE3) as the expression host and then were purified. The purified UGPases were examined using SDS-PAGE (Figure 2A). Approximately 20–45 µg of purified various UGPases was loaded on the gels. The molecular weights of 11 UGPases presented in SDS-PAGE are consistent with their predicted molecular weights in Table 1. The molecular mass of prokaryotic EcUGP is smaller than that of eukaryotic UGPases. The specific activities of 11 UGPases were determined (Figure 2B). Cassava MeUGP and potato StUGP show the highest and second-highest specific activity of 927.1 U/mg and 828.5 U/mg, respectively. EcUGP and ScUGP show the lowest and second-lowest specific activities of 8.2 U/mg and 10.7 U/mg, respectively. Sweet potato IbUGP and maize ZmUGP show specific activities of 159.6 U/mg and 258.2 U/mg, respectively. Cassavas and potatoes are high in starch. Fresh cassavas and potatoes contain ~20% dry matter, of which 60–80% is starch [37,38]. Two UGPases from highly starch-producing plants have the highest specific activity, while the two fungal UGPases have the lowest.

The temperature and pH optima of 11 recombinant UGPases were assayed (Figure 3A). UGPase activity was monitored in different assay temperatures, ranging from 20°C to 80°C. EcUGP, ScUGP, AnUGP, HvUGP, AtUGP, DmUGP, and HsUGP show a temperature optimum of 37°C. MeUGP, IbUGP, and ZmUGP showed a temperature optimum of 50°C. All three UGPases showing higher optimal temperatures are UGPases from plants (Figure 3A). Eleven UGPase activity was monitored in different assay pHs (Figure 3B). Most UGPases had a relatively broad pH optimum between pH 5.0 and 10 and had a sharp drop in activity above pH11 and below pH4 (Figure 3B). For example, StUGP, MeUGP, IBUGP, and ZmUGP retained over 80% activity at pH4–10; ScUGP, DmUGP, and HsUGP retained over 60% activity at pH5–10. The only exception is EcUGP, which had the highest activity at pH7 but rapidly loses activity as pH rises or falls (Figure 3B). The earlier study on barley UGPase also gave a relatively broad optimal pH (5.5–9.5) [32]. The results indicate that plant UGPases have a broad pH adaptation even though plant cell cytosol has a relatively narrow pH range of 6.5–7.3 [39].

The thermal stability of UGPases was monitored in different assay temperatures: 37°C, 50°C, 60°C, and 70°C at pH7.0. Overall, recombinant UGPases were not thermally stable. Only IbUGP retained over 60% of its activity at 60°C for 30 min; the other ten UGPases were rapidly inactivated at 60°C. StUGP, MeUGP, and IbUGP retained most activities at 50°C for 2 h, whereas EcUGP, ScUGP, AnUGP, DmUGP, and HsUGP failed to retain more than 30% activity at 50°C for 30 min. In particular, EcUGP and HsUGP showed a loss of enzyme activity after 2 h at 37°C (Figure 4).

The substrate preferences of six recombinant UGPases for different sugar phosphates and nucleotide triphosphates were compared. Six recombinant UGPases were specific for Glc1P. For example, the activities of MeUGP and StUGP in the presence of other sugar phosphates (Gla1P, GlcA1P, or GlcNAc1P) were less than 10% of those measured in the presence of Glc1P (Figure 5A). The results are consistent with that reported in the plants A. thaliana UGPase and barley UGPase [40]. MeUGP and StUGP used UTP with high activity levels. MeUGP and StUGP can also use other nucleotide triphosphates (CTP, ATP, or GTP) (Figure 5B). AnUGP also used UTP with high activity levels but could not use ATP. Eukaryotic UGPases exhibit wide variations in NTP utilization efficiency. A. thaliana UGP uses UTP as the only effective nucleotide donor, while ScUGP and bovine UGPase can also use ATP as donor substrate, with the relative activities 26–36% of UTP; HvUGP also uses ATP, CTP, and GTP at low relative activities [40,41]. The preference for various substrates should be ascribed to the different structures of various UGPases.

The Michaelis–Menten kinetic parameters k_cat and K_m of 11 recombinant UGPases were determined using different concentrations of Glc1P as the substrate (Table 2). The kinetic curves are shown in Figure 6. The top five second-order rate constant k_cat/K_m values were observed in UGPases from plants (MeUGP, StUGP, ZmUGP, IbUGP, and AtUGP). MeUGP showed the highest k_cat/K value, about 660-fold and 257-fold higher than the lowest EcUGP and the second-lowest ScUGP. StUGP showed the second-highest k_cat/K value, about 181-fold and 70-fold higher than the lowest EcUGP and second-lowest ScUGP. Compared with several UGPases from plants, the k_cat/K values of two UGPases from animals (DmUGP and HsUGP) are also relatively low. StUGPand MeUGP have the highest and second-highest k_cat values, whereas EcUGP and ScUGP have the lowest and second-lowest k_cat values (Table 2).

Among the 11 recombinant UGPases, most UGPases from plants showed higher specific activity and k_cat/K values than those of UGPases from E. coli, budding yeast, A. niger, ScUGP, fruit fly, and human. This is an interesting phenomenon. The analysis of UGPase’s function must consider the enzyme’s product. UDP-Glc is an essential metabolite for the metabolism of carbohydrates in both photosynthetic and nonphotosynthetic tissues [42]. Among the six plant UGPases examined in this study, cassava MeUGP and potato StUGP are the two most active UGPases. Sweet potato IbUGP also exhibits high activity. They are high producers of starch-producing plants. Although ADP-glucose, synthesized by ADP-GPase, is the glucosyl donor for the elongation of alpha-1,4-glucosidic chains in plant starch synthesis, UGPases are closely related to starch accumulation. For example, with 70% less UGPase activity, the rice UGPase1 mutant demonstrated a reduction in starch content along with changes in starch properties of pasting and swelling [43], which may be related to the UGPase-derived Glc1P implicated in starch formation. Based on transcriptome sequencing data of storage root from sweet potato, sugar–starch conversion steps catalyzed by sucrose synthase and UGPase may be essential for starch accumulation [44].

In addition to its role in Suc pathways, UDP-Glc plays a crucial role, either directly or indirectly, in the biosynthesis of cell wall polysaccharides. The overexpression of the UGPase gene in jute (Corchorus capsularis L.) led to increased cellulose content compared with the control, while the lignin content remained unchanged [45]. Similarly, the overexpression of Larix gmelinii (dahurian Larch) UGPase gene resulted in enhanced vegetative growth in transgenic Arabidopsis and increased the contents of soluble sugars and cellulose and thickened parenchyma cell walls [46]. On the other hand, the A. thaliana UGPase1/UGPase2 double mutant displayed drastic growth defects, with absent callose deposition around microspores [19]. Thus, plants might need more active UGPases than microorganisms or animals to produce large quantities of UDP-Glc to support starch accumulation and the production of cell wall polysaccharides such as cellulose, hemicellulose, callose, and pectin.

Changes in the structure of UGPase have been suggested to play an essential role in regulating enzyme activity [20]. Despite structural similarities in the monomers of plant UGPase and other eukaryotic (human and yeast) UGPases, the oligomerization mold of plant UGPase with other eukaryotic UGPases is different. The monomer or mixture of monomer and dimer is the active form of plant UGPase [30,47], whereas octamers are an active form of human and yeast UGPases [12,24]. There may also be oligomerization differences between plant UGPases. A. thaliana UGPase forms a ‘head-to-toe’ dimer, where the N-terminal domain of one monomer faces the C-terminal domain of the other [30]. In contrast, sugarcane UGPase forms a ‘toe-to-toe’ dimer, where the C-terminal domain of the monomers interact with each other, similar to the arrangement of subunits in human and yeast UGPase [48]. Our study showed a high activity of MeUGP and StUGP. The StUGP protein structure remains still unknown, although there have been early reports of StUGP characterization [21,22]. Future research into the structures of StUGP and MeUGP may explain their high catalytic activity.

AnUGP was uncharacterized previously. The UGPases from filamentous fungi have yet to be extensively studied. Only a few reports addressed the transcriptional level of the UGPase gene and UGPase’s role in the mycelial development and virulence of pathogenic fungi [49,50]. The UGPase from model Aspergillus nidulans (AnidUGP) was not characterized until last year [51]. The specific activity of AnidUGP is low (0.41 U/mg). In contrast with other eukaryotic UGPases, AnidUGP displays a few structural variations [52]. The oligomerization mold of AnUGP compared with plant UGPases is different. AnidUGP displays intact octamers, which is also the typical active form of human and yeast UGPases [12,24]. In contrast, the monomer or mixture of monomers and dimer is an active form of plant UGPases [30,47]. Furthermore, despite AnidUGP exhibiting octamers as well as ScUGP and HsUGP, the AnidUGP cryoEM maps do not show proper four-fold symmetry [51]. In contrast, ScUGP and HsUGP show almost perfect four-fold symmetry in the crystal lattice [12,52]. There is a 96.4% similarity between AnUGP and AnidUGP. It is, therefore, speculated that AnUGP may share structural similarities with AnidUGP. Structural differences may account for AnUGP’s lower activity than other eukaryotic UGPases.

UDP-glucose, as the starting point for the synthesis of UDP-sugars (UDP-galactose and UDP-glucuronic acid), is a prerequisite for the rapid synthesis of any oligosaccharides containing these sugars, as well as other related compounds [53]. Thus, UGPases have been used as targets for metabolic engineering to improve the synthesis of products such as glycoglycerolipids, trehalose, hyperoside (quercetin 3-O-galactoside), rhodiola rosea, validatmycin A, and chondroitin-like capsular polysaccharide [54-59] or to produce specific products with unique properties like high molecular weight (>1 MDa) of hyaluronic acid [60,61]. As a target for metabolic engineering, E. coli UGPase was primarily utilized in the studies above [54-59]. Given that UGPases from cassava and potato can be correctly expressed in E. coli and are more active than EcUGP, the results provide the potential to use MeUGP or StUGP as the engineering target of cell factories instead of EcUGP.

Overall, recombinant UGPases were not thermally stable. Among 11 UGPases, ten UGPases were rapidly inactivated at 60°C, except for IbUGP. In particular, EcUGP and HsUGP showed a loss of activity after 2 h at 37°C. The results are unexpected, as 37°C is both the ideal growth temperature for E. coli and the typical body temperature for humans. It is still uncertain whether the buffer in vitro is unsuitable for maintaining UGPase activity. Our studies have shown that the optimal temperature for most recombinant UGPases is 37°C, which is consistent with findings for UGPases from other species such as Streptococcus equi, Caenorhabditis elegans, and Danio rerio [31,62]. The optimal temperature for most UGPases is known to be 21–37°C, which also suggests the instability of UGPases. It is known that a thermostable UGPase with the highest optimal temperature (80°C) is from thermophilic archaeon Sulfolobus tokodaiibacteriafrom [17]. The UGPase from soil actinobacterium Thermocrispum agreste was reported thermostable [63]. It is worthwhile to conduct further research to obtain highly active and stable UGPases to obtain UDP-glucose in vitro.

Using the same assay method, the activities of 11 UGPases from bacteria, fungi, plants, and animals were compared. Plant UGPases commonly have higher activity than others. The top two with the highest enzyme activity are UGPases from potato and cassava, which also show high k_cat/K value. They can potentially be used as the engineering targets of cell factories.

The data underlying this article are available in the article and in its online supplementary material.

The authors declare no conflict of interest.

This research was funded by the National Key Research and Development Program of China, Grant Number 2022YFC3401700 and the National Natural Sciences Foundation of China, Grant Number 32370075 and 32070077.

X.S. and Y.Q.: Conceptualization. S.Z. and Y.Q: Methodology. X.S. and Y.Q.: Validation. Y.Q.: Formal analysis. S.Z. and Y.Q.: Investigation. S.Z. and Y.Q.: Data curation. S.Z. and Y.Q.: Writing—original draft preparation. Y.Q.: Writing—review and editing.X.S. and Y.Q.: Supervision. X.S. and Y.Q.: Project administration. X.S. and Y.Q.: Funding acquisition. All authors have read and agreed to the published version of the manuscript.

AnUGP

Aspergillus niger UGPase

AtUGP

Arabidopsis thaliana UGPase

DmUGP

Drosophila melanogaster UGPase

EcUGP

Escherichia coli UGPase

HsUGP

Homo sapiens UGPase

HvUGP

Hordeum vulgare UGPase

IbUGP

Ipomoea batatas UGPase

MeUGP

Manihot esculenta UGPase

PPi

pyrophosphate

ScUGP

Saccharomyces cerevisiae UGPase

StUGP

Solanum tuberosum UGPase

UGPases

UDP-glucose pyrophosphorylases

ZmUGP

Zea mays UGPase