UDP Sugars Research Articles

Increased sugar content impairs pollen fertility and reduces seed-setting in high-photosynthetic-efficiency rice

Crop yield depends on biomass, which is primarily associated with photosynthesis. We previously demonstrated that two photorespiratory bypasses, i.e., GOC (glycolate oxidase + oxalate oxidase + catalase) and GCGT (glycolate oxidase + catalase + glyoxylate carboligase + tartronic semialdehyde reductase), significantly increased photosynthesis, biomass, and grain yield, but decreased seed-setting rates in rice. This study explored the underlying mechanism of how elevated photosynthetic efficiency impacted the seed-setting. First, pollen germination assessed in vivo and in vitro, revealed a reduced germination rate in GCGT rice. Subsequent analysis found that photosynthates highly accumulated in the leaves and stems; sucrose and soluble sugar levels were increased but the starch level was reduced in the anthers. Uridine diphosphate glucose (UDP-Glc) was increased but uridine diphosphate galactose (UDP-Gal) was unaltered, thus causing an imbalance in the UDP-Glc/UDP-Gal ratio in GCGT anthers. Most anthers in GCGT plants had two locules in contrast to four in the wild-type (WT). Pollen tapetum was developmentally abnormal, and genes related to sucrose synthesis, transport, and tapetal programmed cell death (PCD) were upregulated, whereas those involved in starch synthesis and conversion were downregulated in GCGT anthers. Taken together, our results demonstrated that an increase in sugar content was the primary factor causing reduced seed-setting rates in high photosynthetic efficiency rice, during which metabolic disorder of sugars and UDP sugar imbalance in anthers lead to impaired pollen fertility.

Comparative analysis of differential gene expression reveals novel insights into the heteroblastic foliage functional traits of Pinus massoniana seedlings

Pinus massoniana needles, rich in medicinal polysaccharides and flavonoids, undergo heteroblastic foliage, transitioning from primary needles (PN) to secondary needles (SN) during growth, resulting in altered functional traits. Despite its significance, the molecular regulatory mechanisms governing these traits remain unclear. This study employs Iso-Seq and RNA-Seq analyses to explore differentially expressed genes (DEGs) associated with functional traits throughout the main growth season of heteroblastic foliage. Co-expression network analysis identified 34 hub genes and 17 key transcription factors (TFs) influencing light-harvesting antenna, photosystem I and II, crucial in photosynthesis regulation. Additionally, 14 genes involved in polysaccharide metabolism pathways, synthesizing sucrose, glucose, UDP sugars, and xylan, along with four genes in flavonoid biosynthesis pathways, regulating p-coumaroyl-CoA, quercetin, galangin, and myricetin production, exhibited differential expression between PN and SN. Further analysis unveils a highly interconnected network among these genes, forming a pivotal cascade of TFs and DEGs. Therefore, heteroblastic changes significantly impact needle functional traits, potentially affecting the pharmacological properties of PN and SN. Thus, these genomic insights into understanding the molecular-level differences of heteroblastic foliage, thereby establishing a foundation for advancements in the pharmaceutical industry related to needle-derived products.

Long read transcriptome sequencing of a sugarcane hybrid and its progenitors, Saccharum officinarum and S. spontaneum.

Commercial sugarcane hybrids are derivatives from Saccharum officinarum and Saccharum spontaneum hybrids containing the full complement of S. officinarum and a few S. spontaneum chromosomes and recombinants with favorable agronomic characters from both the species. The combination of the two sub-genomes in varying proportions in addition to the recombinants presents a challenge in the study of gene expression and regulation in the hybrid. We now report the transcriptome analysis of the two progenitor species and a modern commercial sugarcane hybrid through long read sequencing technology. Transcripts were profiled in the two progenitor species S. officinarum (Black Cheribon), and S. spontaneum (Coimbatore accession) and a recent high yielding, high sugar variety Co 11015. The composition and contribution of the progenitors to a hybrid with respect to sugar, biomass, and disease resistance were established. Sugar related transcripts originated from S. officinarum while several stress and senescence related transcripts were from S. spontaneum in the hybrid. The hybrid had a higher number of transcripts related to sugar transporters, invertases, transcription factors, trehalose, UDP sugars, and cellulose than the two progenitor species. Both S. officinarum and the hybrid had an abundance of novel genes like sugar phosphate translocator, while S. spontaneum had just one. In general, the hybrid shared a larger number of transcripts with S. officinarum than with S. spontaneum, reflecting the genomic contribution, while the progenitors shared very few transcripts between them. The common isoforms among the three genotypes and unique isoforms specific to each genotype indicate that there is a high scope for improvement of the modern hybrids by utilizing novel gene isoforms from the progenitor species.

Characterization of UDP-glycosyltransferase family members reveals how major flavonoid glycoside accumulates in the roots of Scutellaria baicalensis

BackgroundFlavonoid glycosides extracted from roots of Scutellaria baicalensis exhibit strong pharmaceutical antitumor, antioxidative, anti-inflammatory, and antiviral activities. UDP glycosyltransferase (UGT) family members are responsible for the transfer of a glycosyl moiety from UDP sugars to a wide range of acceptor flavonoids. Baicalin is the major flavonoid glycoside found in S. baicalensis roots, and its aglycone baicalein is synthesized from a specially evolved pathway that has been elucidated. However, it is necessary to carry out a genome-wide study of genes involved in 7-O-glucuronidation, the final biosynthesis step of baicalin, which might elucidate the relationship between the enzymes and the metabolic accumulation patterns in this medicinal plant.ResultsWe reported the phylogenetic analysis, tissue-specific expression, biochemical characterization and evolutionary analysis of glucosyltransferases (SbUGTs) and glucuronosyltransferases (SbUGATs) genes based on the recently released genome of S. baicalensis. A total of 124 UGTs were identified, and over one third of them were highly expressed in roots. In vitro enzyme assays showed that 6 SbUGTs could use UDP-glucose as a sugar donor and convert baicalein to oroxin A (baicalein 7-O-glucoside), while 4 SbUGATs used only UDP-glucuronic acid as the sugar donor and catalyzed baicalein to baicalin. SbUGAT4 and SbUGT2 are the most highly expressed SbUGAT and SbUGT genes in root tissues, respectively. Kinetic measurements revealed that SbUGAT4 had a lower Km value and higher Vmax/Km ratio to baicalein than those of SbUGT2. Furthermore, tandem duplication events were detected in SbUGTs and SbUGATs.ConclusionsThis study demonstrated that glucosylation and glucuronidation are two major glycosylated decorations in the roots of S. baicalensis. Higher expression level and affinity to substrate of SbUGAT4, and expansion of this gene family contribute high accumulation of baicalin in the root of S. baicalensis.

The Antifungal Effects of Citral on Magnaporthe oryzae Occur via Modulation of Chitin Content as Revealed by RNA-Seq Analysis.

The natural product citral has previously been demonstrated to possess antifungal activity against Magnaporthe oryzae. The purpose of this study was to screen and annotate genes that were differentially expressed (DEGs) in M. oryzae after treatment with citral using RNA sequencing (RNA-seq). Thereafter, samples were reprepared for quantitative real-time PCR (RT-qPCR) analysis verification of RNA-seq data. The results showed that 649 DEGs in M. oryzae were significantly affected after treatment with citral (100 μg/mL) for 24 h. Kyoto Encyclopedia of Genes and Genomes (KEGG) and a gene ontology (GO) analysis showed that DEGs were mainly enriched in amino sugar and nucleotide sugar metabolic pathways, including the chitin synthesis pathway and UDP sugar synthesis pathway. The results of the RT-qPCR analysis also showed that the chitin present in M. oryzae might be degraded to chitosan, chitobiose, N-acetyl-D-glucosamine, and β-D-fructose-6-phosphate following treatment with citral. Chitin degradation was indicated by damaged cell-wall integrity. Moreover, the UDP glucose synthesis pathway was involved in glycolysis and gluconeogenesis, providing precursors for the synthesis of polysaccharides. Galactose-1-phosphate uridylyltransferase, which is involved in the regulation of UDP-α-D-galactose and α-D-galactose-1-phosphate, was downregulated. This would result in the inhibition of UDP glucose (UDP-Glc) synthesis, a reduction in cell-wall glucan content, and the destruction of cell-wall integrity.

Identification and characterization of AcUFGT6b, a xylosyltransferase involved in anthocyanin modification in red-fleshed kiwifruit (Actinidia chinensis)

Anthocyanins are responsible for the red color in red-fleshed kiwifruits. In most of these cyanidin 3-O-xyl-galactoside is the predominant anthocyanin. This requires a second glycosylation modification which attaches the UDP-xylose to the cyanidin 3-O-galactoside. We report here the functional characterization of two second glycosyltransferases from ‘Hongyang’ kiwifruit, AcUFGT6b and AcUFGT7c. These were found to cluster with other plant GGTs. In vitro, recombinant AcUFGT6b recognized 3-O-glycosylated anthocyanins and UDP-xylose, while AcUFGT7c showed no or negligible activity. In tobacco leaves, co-expression of AcMYBF110, AcUFGT3a and AcUFGT6b resulted in the accumulation of three new products: cyanidin 3-O-xyl-galactoside, cyanidin 3-O-xyl-glucoside and cyanidin 3-O-xyl-rutoside. RNAi assays showed that a lack of AcUFGT6b and AcUFGT7c in immature fruits of Actinidia arguta prevents the formation of cyanidin 3-O-xyl-galactoside. Also, the content of cyanidin 3-O-xyl-galactoside was obviously increased when AcUFGT6b was injected together with AcMYBF110 and AcUFGT6b7c-RNAi. In contrast, for AcUFGT7c, as well as in vitro, we show no activity in either tobacco leaves or kiwifruit. Both AcUFGT6b and AcUFGT7c are located in the ER and their promoters can be positively activated by AcMYBF110, indicating the functional differences of them are due neither to their cell localization nor to the upstream regulation of AcMYBF110. Interestingly, the deletion in the C-terminal of AcUFGT7c is likely to change its 3D protein structure compared with AcUFGT6b protein, resulting in it having no capacity to attract the UDP sugar moiety. These results suggest that AcUFGT6b, but not AcUFGT7c, is responsible for the end-product of the anthocyanins biosynthesis pathway in red-fleshed kiwifruit.

Heparin affects cytosolic glucose responses of hyperglycemic dividing mesangial cells

Mesangial expansion underlies diabetic nephropathy, leading to sclerosis and renal failure. The glycosaminoglycan heparin inhibits mesangial cell growth, but the molecular mechanism is unclear. Here, rat mesangial cells (RMCs) were growth-arrested in the G0/G1 phase of cell division, stimulated to divide in normal glucose (5.6 mm) or high glucose (25.6 mm) with or without heparin, and analyzed for glucose uptake. We observed that RMCs entering the G1 phase in normal glucose with or without heparin rapidly cease glucose uptake. RMCs entering G1 in high glucose sustained glucose uptake for the first 3 h, and high-glucose exposure of RMCs only in the first 8 h of G1 induced the formation of an extracellular monocyte-adhesive hyaluronan matrix after cell division was completed. Moreover, a low heparin concentration under high-glucose conditions blocked glucose uptake by 1 h into G1 Of note, glucose transporter 4 (glut4) localized on the RMC surface at G0/G1 and was internalized into G1 cells under normal glucose conditions with or without heparin within 30 min. We also noted that, under high-glucose conditions, glut4 remained on the RMC surface for at least 2 h into G1 and was internalized by 4 h without heparin and within 1 h with heparin. These results provide evidence that the influx of glucose in hyperglycemic dividing RMCs initiates intermediate glucose metabolism, leading to increased cytosolic UDP sugars, and induces abnormal intracellular hyaluronan synthesis during the S phase of cell division.

Structural determination of archaeal UDP-N-acetylglucosamine 4-epimerase from Methanobrevibacter ruminantium M1 in complex with the bacterial cell wall intermediate UDP-N-acetylmuramic acid.

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.

Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases.

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 Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP-N-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 (Km 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 (Km of 1.3 mM), β-L-Ara-1-P and α-D-Fuc-1-P (Km 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 (Km of 1 mM) and, to some extent, D-Glc-1-P (Km 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.

Synthesis of 4-Azido-N-acetylhexosamine Uridine Diphosphate Donors: Clickable Glycosaminoglycans.

Unnatural chemically modified nucleotide sugars UDP-4-N3-GlcNAc and UDP-4-N3-GalNAc were chemically synthesized for the first time. These unnatural UDP sugar products were then tested for incorporation into hyaluronan, heparosan, or chondroitin using polysaccharide synthases. UDP-4-N3-GlcNAc served as a chain termination substrate for hyaluronan or heparosan synthases; the resulting 4-N3-GlcNAc-terminated hyaluronan and heparosan were then successfully conjugated with Alexa Fluor 488 DIBO alkyne, demonstrating that this approach is generally applicable for labeling and detection of suitable glycosaminoglycans.

Abstract C41: Altered control of intracellular androgen availability by glucuronidation contributes to loss of androgen dependence in prostate tumor cells

Abstract Locally advanced and metastatic cancers are frequently treated by androgen deprivation therapy. Tumors that recur despite this treatment, termed castration resistant prostate cancer, are highly aggressive and have acquired the capacity to survive and expand in conditions of low circulating androgens. Prostate epithelial cells control the potency and overall availability of androgen hormones in part by inactivation and excretion of excess quantities. The enzymes that catalyze inactivation, specifically by covalently modifying the hormone with a glucuronate adduct, are called UDP-glucuronosyltransferases (UGT). Genetic mutations that reduce levels of the UGT isozymes needed to eliminate excess androgens from the prostate have been correlated with prostate cancer incidence. The ability to alter androgen elimination at the level of individual cells within a tumor is one mechanism by which tumors become heterogeneous with respect to molecular features and therapeutic resistance. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD+-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. Our laboratory has found that non-metastatic prostate tumor cell lines express UGDH in an androgen sensitive manner, that its expression increases the level of UDP-glucuronate, and that these precursors are then channeled to the UGT-catalyzed glucuronidation pathway. The level of UGDH is androgen controlled and serves to regulate tumor cell growth, inversely to its expression. Loss of androgen stimulated UGDH, or pharmacological antagonism of its activity, translated to increased proliferation rate, specifically of androgen dependent tumor cells. We knocked down expression of UGDH in androgen dependent cells to test the role of UGDH in driving cellular glucuronate levels, intratumoral steroid availability, and response to androgen. Flux of UDP sugar metabolites and levels of androgen glucuronide accumulation were compared in control and UGDH knockdown lines by liquid chromatography and mass spectrometry analysis. Effects on androgen sensitive gene expression were monitored by western analysis. Selection of manipulated cells in decremental DHT levels that simulated androgen deprivation was found to reduce steady state UGDH expression, thereby activating the UGT-driven steroid clearance mechanism, as reflected in increased PSA levels and de-repression of UGT2B17. Whereas restimulation of control transfectants with physiological levels of DHT could only increase UGDH expression and maintain full androgen sensitivity in cells experiencing complete androgen loss, the knockdown of UGDH permitted cells to accumulate UDP-glucuronate pools, and decreased UGT-driven glucuronidation of DHT and androsterone. These results support a model in which maintenance of partial UGDH activity through gradual androgen deprivation facilitates loss of sensitivity in androgen elimination pathways and suggests adjuvant UGDH inhibition could prevent or delay onset of castration resistant recurrence. Citation Format: Michelle Palmer, Linlin Ma, Trevor Romsdahl, Qin Wei, Eileen Loughman, Jonathan Markham, Joseph Barycki, Melanie A. Simpson. Altered control of intracellular androgen availability by glucuronidation contributes to loss of androgen dependence in prostate tumor cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C41.

YZGD, a Two‐Domain Enzyme, as a Teaching Tool in a Biochemistry Laboratory

YZGD is a two domain enzyme that is both a member of the HAD superfamily with pyridoxal phosphatase activity and a member of the Nudix Hydrolase superfamily with comparable activity on CDP alcohols, ADP sugars and ADP coenzymes, TDP sugars, and some activity on UDP and GDP sugars. Due to its two‐domain nature, I have found it to be a useful enzyme to use in the teaching laboratory. We have created a mutant (YZGDD12N) that has no pyridoxal phosphatase activity but full Nudix activity and a mutant (YZGDE324Q) that has no Nudix activity but full pyridoxal phosphatase activity, demonstrating the independent nature of enzyme domains. We also have cloned both a his‐tagged YZGD and a non‐his‐tagged YZGD, thus demonstrating the power of affinity chromatography in enzyme purification. The students express the various YZGDs (mutants, wildtype, and his‐tagged), purify these enzymes, and characterize them (Bradford assays, gel electrophoresis, and enzyme assays). We then work as a group to compare and contrast the results that each student gets; it is this comparison that especially shows the students the power of site‐directed mutagenesis and affinity chromatography purification. In addition, the students do a protein structure ‐ function project where they use the structural database SCOP, the visualization program CN3D, BLAST searches and CLUSTAL alignments, and the scientific literature on any HAD or Nudix superfamily member that has been structurally determined.

UDP-Sugars as Extracellular Signaling Molecules: Cellular and Physiologic Consequences of P2Y14 Receptor Activation.

UDP-sugars, which are indispensable for protein glycosylation reactions in cellular secretory pathways, also act as important extracellular signaling molecules. We discuss here the broadly expressed P2Y14 receptor, a G-protein-coupled receptor targeted by UDP sugars, and the increasingly diverse set of physiologic responses discovered recently functioning downstream of this receptor in many epithelia as well as in immune, inflammatory, and other cells.

The G Protein-coupled Receptor P2Y14 Influences Insulin Release and Smooth Muscle Function in Mice

UDP sugars were identified as extracellular signaling molecules, assigning a new function to these compounds in addition to their well defined role in intracellular substrate metabolism and storage. Previously regarded as an orphan receptor, the G protein-coupled receptor P2Y14 (GPR105) was found to bind extracellular UDP and UDP sugars. Little is known about the physiological functions of this G protein-coupled receptor. To study its physiological role, we used a gene-deficient mouse strain expressing the bacterial LacZ reporter gene to monitor the physiological expression pattern of P2Y14. We found that P2Y14 is mainly expressed in pancreas and salivary glands and in subpopulations of smooth muscle cells of the gastrointestinal tract, blood vessels, lung, and uterus. Among other phenotypical differences, knock-out mice showed a significantly impaired glucose tolerance following oral and intraperitoneal glucose application. An unchanged insulin tolerance suggested altered pancreatic islet function. Transcriptome analysis of pancreatic islets showed that P2Y14 deficiency significantly changed expression of components involved in insulin secretion. Insulin secretion tests revealed a reduced insulin release from P2Y14-deficient islets, highlighting P2Y14 as a new modulator of proper insulin secretion.

Hyaluronan Produced by Smooth Muscle Cells Plays a Critical Role in Neointima Formation

Large body of evidence supports the idea that microenvironment plays a critical role in several pathologies including atherosclerosis and cancer. The amount of hyaluronan (HA) is involved in the microenvironment alterations and the concentration of this polymer reflects the progression of the diseases promoting neoangiogenesis, cell migration, and inflammation. The HA synthesis is regulated by several factors: UDP sugar precursors availability and the phosphorylation of synthetic enzyme HAS2 as well as specific drugs reducing the UDP precursors. The HAS2 phosphorylation is done by AMP kinase, a sensor of cell energy. When the cells have low energy, AMP kinase is activated and modifies covalently the regulatory enzymes, blocking all biosynthetic processes and activating the energy producing metabolism. It was recently reported that the hexosamine biosynthetic pathway (HBP) may increase the concentration of HA precursor UDP-N-acetylglucosamine (UDP-GlcNAc) leading to an increase of HA synthesis. We demonstrated that the increase of HA synthesis depends on the HAS2 post translational modification O-GlcNAcylation, which increases HA secretion modifying a residue different from the phosphorylation site of AMP kinase. In this report we highlighted the critical aspects of the post translational HAS2 regulation and its influence on HA synthesis.

Kinetic and structural analysis of two transferase domains in Pasteurella multocida hyaluronan synthase

Abstract Pasteurella multocida hyaluronan synthase (PmHAS) encompasses two transferase domains that elongate a growing hyaluronan (HA) oligosaccharide chain by addition of either GlcNAc or GlcUA residues from a corresponding UDP-sugar. Initial velocity studies of single-step elongations were conducted for both domains by independently varying the concentrations of the HA oligosaccharide and the UDP-sugar. Two-substrate models were discriminated by their goodness-of-fit parameters and by dead-end inhibition studies. A mechanistic shift from a steady-state ordered bi-bi to rapid equilibrium ordered bi-bi mechanism was observed at the NAc-site between the HA6 and HA8 elongation. This shift was invoked by a minor reduction in turnover number kcat. Both NAc- and UA-transferase domains follow a sequential kinetic mechanism, most likely an ordered one in which the UDP-sugar donor binds first, followed by the HA oligosaccharide. After transfer of the sugar moiety, both products are released, first the elongated HA oligosaccharide and then the UDP sugar. This mechanism was visualized with a structural model of PmHAS that presented two flexible loops, one in each transferase domain; these loops form a bridge above the active site. Based on structural similarities between PmHAS with α1,3-galactosyltransferase (α3GT) and β1,4-galactosyltransferase (β4Gal-T1), these flexible loops are seemingly involved in a conformational change upon binding the UDP-sugar, inducing the ordered mechanism. Kinetic analyses have demonstrated significant differences in dissociation constant KD,UA and KM,NAc of 124 ± 31 μM and 28 ± 8.8 mM, respectively, suggesting that the variance in the binding affinity of the two UDP-sugars controls the molecular weight of hyaluronan.

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in k(cat)/K(m) of >400-fold and >15-fold for formation of NDP-glucoses and UDP-sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP-sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.

Genome-Wide mRNA and microRNA Profiling of the NCI 60 Cell-Line Screen and Comparison of FdUMP[10] with Fluorouracil, Floxuridine, and Topoisomerase 1 Poisons

A profile of microRNA (miRNA) and mRNA expression patterns across the NCI-60 cell-line screen was analyzed to identify expression signatures that correlate with sensitivity to FdUMP[10], fluorouracil (5FU), floxuridine (FdU), topotecan, and irinotecan. Genome-wide profile analyses revealed FdUMP[10] resembles FdU most closely and shows dissimilarities with 5FU. FdUMP[10] had the largest dynamic range of any of these drugs across the NCI-60 indicative of cancer cell-specific activity. Genes involved in endocytosis, such as clathrin (CLTC), SNF8, annexin A6 (ANXA6), and amyloid protein-binding 2 (APPBP2) uniquely correlated with sensitivity to FdUMP[10], consistent with a protein-mediated cellular uptake of FdUMP[10]. Genes involved in nucleotide metabolism were enriched for the three fluoropyrimidine drugs, with the expression profile for 5FU correlated to an RNA-mediated cytotoxic mechanism, whereas expression of glycosyltransferases (XYLT2) that use UDP sugars as substrates and the nucleoside diphosphatase and metastasis suppressor NM23 (NME1) were associated with FdUMP[10] sensitivity. Topotecan and irinotecan had significant negative correlations with miR-24, a miRNA with a high aggregate P(CT) score for topoisomerase 1 (Top1). Our results reveal significant new correlations between FdUMP[10] and Top1 poisons, as well as new information on the unique cytotoxic mechanism and genomic signature of FdUMP[10].

The Novel UDP Glycosyltransferase 3A2: Cloning, Catalytic Properties, and Tissue Distribution

The human UDP glycosyltransferase (UGT) 3A family is one of three families involved in the metabolism of small lipophilic compounds. Members of these families catalyze the addition of sugar residues to chemicals, which enhances their excretion from the body. The UGT1 and UGT2 family members primarily use UDP glucuronic acid to glucuronidate numerous compounds, such as steroids, bile acids, and therapeutic drugs. We showed recently that UGT3A1, the first member of the UGT3 family to be characterized, is unusual in using UDP N-acetylglucosamine as sugar donor, rather than UDP glucuronic acid or other UDP sugar nucleotides (J Biol Chem 283:36205-36210, 2008). Here, we report the cloning, expression, and characterization of UGT3A2, the second member of the UGT3 family. Like UGT3A1, UGT3A2 is inactive with UDP glucuronic acid as sugar donor. However, in contrast to UGT3A1, UGT3A2 uses both UDP glucose and UDP xylose but not UDP N-acetylglucosamine to glycosidate a broad range of substrates including 4-methylumbelliferone, 1-hydroxypyrene, bioflavones, and estrogens. It has low activity toward bile acids and androgens. UGT3A2 transcripts are found in the thymus, testis, and kidney but are barely detectable in the liver and gastrointestinal tract. The low expression of UGT3A2 in the latter, which are the main organs of drug metabolism, suggests that UGT3A2 has a more selective role in protecting the organs in which it is expressed against toxic insult rather than a more generalized role in drug metabolism. The broad substrate and novel UDP sugar specificity of UGT3A2 would be advantageous for such a function.

Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum

Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP–sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a coupling enzyme. Glucuronokinase was partially purified on a DEAE–sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is used by UDP–sugar pyrophosphorylase and further converted to UDP–glucuronic acid. This UDP–sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange column or a reversed phase C18 column at a wavelength of 260 nm. This assay is adaptive to different kinases and sugars because of the broad substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min –1. Furthermore, it can be used for determination of pure kinases as well as crude or partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.