| 1. |
- Decker, Daniel, 1986-, et al.
(author)
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Exploring redox modulation of plant UDP-glucose pyrophosphorylase
- 2023
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In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 24:10
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Journal article (peer-reviewed)abstract
- UDP-glucose (UDPG) pyrophosphorylase (UGPase) catalyzes a reversible reaction, producing UDPG, which serves as an essential precursor for hundreds of glycosyltransferases in all organisms. In this study, activities of purified UGPases from sugarcane and barley were found to be reversibly redox modulated in vitro through oxidation by hydrogen peroxide or oxidized glutathione (GSSG) and through reduction by dithiothreitol or glutathione. Generally, while oxidative treatment decreased UGPase activity, a subsequent reduction restored the activity. The oxidized enzyme had increased Km values with substrates, especially pyrophosphate. The increased Km values were also observed, regardless of redox status, for UGPase cysteine mutants (Cys102Ser and Cys99Ser for sugarcane and barley UGPases, respectively). However, activities and substrate affinities (Kms) of sugarcane Cys102Ser mutant, but not barley Cys99Ser, were still prone to redox modulation. The data suggest that plant UGPase is subject to redox control primarily via changes in the redox status of a single cysteine. Other cysteines may also, to some extent, contribute to UGPase redox status, as seen for sugarcane enzymes. The results are discussed with respect to earlier reported details of redox modulation of eukaryotic UGPases and regarding the structure/function properties of these proteins.
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| 2. |
- Decker, Daniel, 1986-, et al.
(author)
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Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies
- 2017
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In: The Plant Journal. - : John Wiley & Sons. - 0960-7412 .- 1365-313X. ; 90:6, s. 1093-1107
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Journal article (other academic/artistic)abstract
- UDP-sugars serve as ultimate precursors in hundreds of glycosylation reactions (e.g. for protein and lipid glycosylation, synthesis of sucrose, cell wall polysaccharides, etc.), underlying an important role of UDP-sugar-producing enzymes in cellular metabolism. However, genetic studies on mechanisms of UDP-sugar formation were frequently hampered by reproductive impairment of the resulting mutants, making it difficult to assess an in vivo role of a given enzyme. Here, a chemical library containing 17 500 compounds was separately screened against purified UDP-glucose pyrophosphorylase (UGPase) and UDP-sugar pyrophosphorylase (USPase), both enzymes representing the primary mechanisms of UDP-sugar formation. Several compounds have been identified which, at 50 μm, exerted at least 50% inhibition of the pyrophosphorylase activity. In all cases, both UGPase and USPase activities were inhibited, probably reflecting common structural features of active sites of these enzymes. One of these compounds (cmp #6), a salicylamide derivative, was found as effective inhibitor of Arabidopsis pollen germination and Arabidopsis cell culture growth. Hit optimization on cmp #6 yielded two analogs (cmp #6D and cmp #6D2), which acted as uncompetitive inhibitors against both UGPase and USPase, and were strong inhibitors in the pollen test, with apparent inhibition constants of less than 1 μm. Their effects on pollen germination were relieved by addition of UDP-glucose and UDP-galactose, suggesting that the inhibitors targeted UDP-sugar formation. The results suggest that cmp #6 and its analogs may represent useful tools to study in vivo roles of the pyrophosphorylases, helping to overcome the limitations of genetic approaches.
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| 3. |
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| 4. |
- Decker, Daniel, 1986-, et al.
(author)
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The structure-activity relationship of the salicylimide derived inhibitors of UDP-sugar producing pyrophosphorylases
- 2018
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In: Plant Signalling & Behavior. - : Taylor & Francis. - 1559-2316 .- 1559-2324. ; 13:8
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Journal article (peer-reviewed)abstract
- UDP-sugars are key precursors for biomass production in nature (synthesis of cellulose, hemicellulose, etc.). They are produced de novo by distinct UDP-sugar producing pyrophosphorylases. Studies on the roles of these enzymes using genetic knockouts were hampered by sterility of the mutants and by functional-complementation from related enzyme(s), hindering clear interpretation of the results. In an attempt to override these difficulties, we turned to the reverse chemical genetics approaches to identify compounds which interfere with the activity of those enzymes in vivo. Hit expansion on one of such compounds, a salicylimide derivative, allowed us to identify several inhibitors with a range of activities. The present study provides a structure-activity relationship for these compounds.
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| 5. |
- Decker, Daniel, 1986-
(author)
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UDP-sugar metabolizing pyrophosphorylases in plants : formation of precursors for essential glycosylation-reactions
- 2017
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Doctoral thesis (other academic/artistic)abstract
- UDP-sugar metabolizing pyrophosphorylases provide the primary mechanism for de novo synthesis of UDP-sugars, which can then be used for myriads of glycosyltranferase reactions, producing cell wall carbohydrates, sucrose, glycoproteins and glycolipids, as well as many other glycosylated compounds. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase) and UDP-N-acety lglucosamine pyrophosphorylase (UAGPase), which can be discriminated both by differences in accepted substrate range and amino acid sequences.This thesis focuses both on experimental examination (and re-examination) of some enzymatic/ biochemical properties of selected members of the UGPases and USPases and UAGPase families and on the design and implementation of a strategy to study in vivo roles of these pyrophosphorylases using specific inhibitors. In the first part, substrate specificities of members of the Arabidopsis UGPase, USPase and UAGPase families were comprehensively surveyed and kinetically analyzed, with barley UGPase also further studied with regard to itspH dependency, regulation by oligomerization, etc. 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 Frc-1-P, whereas USPase reacted with arange of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P, β-L-Ara-1-P and α-D-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P and, to some extent, with D-Glc-1-P. A structure activity relationship was established to connect enzyme activity, the examined sugar-1-phosphates and the three pyrophosphorylases. The UGPase/USPase/UAGPase active sites were subsequently compared in an attempt to identify amino acids which may contribute to the experimentally determined differences in substrate specificities.The second part of the thesis deals with identification and characterization of inhibitors of the pyrophosphorylases and with studies on in vivo effects of those inhibitors in Arabidopsis-based systems. A novel luminescence-based high-throughput assay system was designed, which allowed for quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The assay was then used to screen a chemical library (which contained 17,500 potential inhibitors) to identify several compounds affecting UGPase and USPase. Hit-optimization on one of the compounds revealed even stronger inhibitors of UGPase and USPase which also strongly inhibited Arabidopsis pollen germination, by disturbing UDP-sugar metabolism. The inhibitors may represent useful tools to study in vivo roles of the pyrophosphorylases, as a complement to previous genetics-based studies.The thesis also includes two review papers on mechanisms of synthesis of NDP-sugars. The first review covered the characterization of USPase from both prokaryotic and eukaryotic organisms, whereas the second review was a comprehensive survey of NDP-sugar producing enzymes (not only UDP-sugar producing and not only pyrophosphorylases). All these enzymes were discussed with respect to their substrate specificities and structural features (if known) and their proposed in vivo functions.
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| 6. |
- Decker, Daniel, 1986-, et al.
(author)
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UDP-Sugar Producing Pyrophosphorylases : Distinct and Essential Enzymes With Overlapping Substrate Specificities, Providing de novo Precursors for Glycosylation Reactions
- 2019
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In: Frontiers in Plant Science. - : Frontiers Media S.A.. - 1664-462X. ; 9
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Research review (peer-reviewed)abstract
- Nucleotide sugars are the key precursors for all glycosylation reactions and are required both for oligo- and polysaccharides synthesis and protein and lipid glycosylation. Among all nucleotide sugars, UDP-sugars are the most important precursors for biomass production in nature (e.g., synthesis of cellulose, hemicellulose, and pectins for cell wall production). Several recent studies have already suggested a potential role for UDP-Glc in plant growth and development, and UDP-Glc has also been suggested as a signaling molecule, in addition to its precursor function. In this review, we will cover primary mechanisms of formation of UDP-sugars, by focusing on UDP-sugar metabolizing pyrophosphorylases. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase), and UDP-N-acetyl glucosamine pyrophosphorylase (UAGPase), which can be distinguished both by their amino acid sequences and by differences in substrate specificity. Substrate specificities of these enzymes are discussed, along with structure-function relationships, based on their crystal structures and homology modeling. Earlier studies with transgenic plants have revealed that each of the pyrophosphorylases is essential for plant survival, and their loss or a decrease in activity results in reproductive impairment. This constitutes a problem when studying exact in vivo roles of the enzymes using classical reverse genetics approaches. Thus, strategies involving the use of specific inhibitors (reverse chemical genetics) are also discussed. Further characterization of the properties/roles of pyrophosphorylases should address fundamental questions dealing with mechanisms and control of carbohydrate synthesis and may allow to identify targets for manipulation of biomass production in plants.
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| 7. |
- Kleczkowski, Leszek A., 1954-, et al.
(author)
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Effects of Magnesium, Pyrophosphate and Phosphonates on Pyrophosphorolytic Reaction of UDP-Glucose Pyrophosphorylase
- 2022
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In: PLANTS. - : MDPI. - 2223-7747. ; 11:12
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Journal article (peer-reviewed)abstract
- UDP-glucose pyrophosphorylase (UGPase) carries a freely reversible reaction, using glucose-1-P and UTP to produce UDP-glucose (UDPG) and pyrophosphate (PPi ), with UDPG being essential for glycosylation reactions in all organisms including, e.g., synthesis of sucrose, cellulose and glycoproteins. In the present study, we found that free magnesium (Mg2+) had profound effects on the reverse reaction of purified barley UGPase, and was absolutely required for its activity, with an apparent Km of 0.13 mM. More detailed analyses with varied concentrations of MgPPi allowed us to conclude that it is the MgPPi complex which serves as true substrate for UGPase in its reverse reaction, with an apparent Km of 0.06 mM. Free PPi was an inhibitor in this reaction. Given the key role of PPi in the UGPase reaction, we have also tested possible effects of phosphonates, which are analogs of PPi and phosphate (Pi ). Clodronate and etidronate (PPi analogs) had little or no effect on UGPase activity, whereas fosetyl-Al (Pi analog), a known fungicide, acted as effective near-competitive inhibitor versus PPi, with Ki of 0.15 mM. The data are discussed with respect to the role of magnesium in the UGPase reaction and elucidating the use of inhibitors in studies on cellular function of UGPase and related enzymes.
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| 8. |
- Kleczkowski, Leszek A., 1954-, et al.
(author)
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Sugar activation for production of nucleotide sugars as aubstrates for glycosyltransferases in plants
- 2015
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In: Journal of Applied Glycoscience. - Tokyo : The Japanese Society of Applied Glycoscience. - 1344-7882 .- 1880-7291. ; 62:2, s. 25-36
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Research review (peer-reviewed)abstract
- In order to serve as a glycosyl donor, a sugar or a sugar derivative (e.g. GlcA) needs to be “activated” to a highly energetic state of a nucleotide-sugar. This activation requires the involvement of specific enzymes which produce NDP-sugars (or, in one case, NMP-sugar), using NTP or NDP as substrate. The present review provides concise survey of distinct plant nucleotide-sugar pyrophosphorylases (all using NTP as one of the substrates and differing in sugar specificity) as well as nucleotide-sugar phosphorylases and sucrose synthase (all using NDP as one of substrates). The pyrophosphorylases discussed include UGPase, USPase, UAGPase, AGPase, GMPase (VTC1), and FKGP, whereas phosphorylases include ADP-Glc phosphorylase and GDP-Gal phosphorylase (VTC2/VTC5). We also discuss the activation mechanism of 3-deoxy-D-manno-octulosonic acid (Kdo) by CKS, leading to the formation of a unique NMP-linked sugar (CMP-Kdo).
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| 9. |
- Kleczkowski, Leszek A, 1954-, et al.
(author)
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UDP-sugar pyrophosphorylase : a new old mechanism for sugar activation
- 2011
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In: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 156:1, s. 3-10
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Journal article (peer-reviewed)abstract
- Recent developments in studies on properties and functions of UDP-sugar pyrophosphorylase (USPase) in metabolism are presented. The protein was characterized from plants and protozoans (Leishmania, Trypanosoma), but apparently it is also present in bacteria. In plants, USPase deficiency leads to male-sterility. USPase produces a variety of UDP-sugars and their analogs required for cell wall biosynthesis as well as for protein and lipid glycosylation, among other functions. Substrate specificity of USPases from different sources is reviewed, and their function/ structure properties are discussed, based on recent crystallization of the protein, with emphasis on common structural blueprint with some other pyrophosphorylases. Some strategies for future research on USPase are discussed.
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