The Trypanosome UDP-Glucose Pyrophosphorylase Is Imported by Piggybacking into Glycosomes, Where Unconventional Sugar Nucleotide Synthesis Takes Place

ABSTRACT Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways, such as glycolysis and biosynthesis of sugar nucleotides, usually present in the cytosol of other eukaryotes. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localized in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisome-targeting signal (PTS1 and PTS2). The questions that we address here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotides functional and (ii) how is the PTS-free UGP imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3 to 10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for the growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knockdown UGP mutant cell line (RNAiUGP, where RNAi indicates RNA interference) was rescued by expressing a recoded UGP (rUGP) in the organelle (RNAiUGP/EXPrUGP-GPDH, where GPDH is glycerol-3-phosphate dehydrogenase). Our conclusion was supported by targeted metabolomic analyses (ion chromatography–high-resolution mass spectrometry [IC-HRMS]) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. IMPORTANCE Unusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localization of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here, we showed that the cytosolic and glycosomal pathways are functional. As in all other eukaryotes, the cytosolic pathways feed glycosylation reactions; however, the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK); they are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

The trypanosome UDP-glucose pyrophosphorylase is imported by piggybacking into glycosomes where unconventional sugar nucleotide synthesis takes place

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways usually present in the cytosol of other eukaryotes, such as glycolysis and biosynthesis of sugar nucleotides. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localised in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisomal targeting signal (PTS1 and PTS2). The questions we addressed here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotide functional and (ii) how the PTS-free UGP is imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3-10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knock-down UGP mutant cell line (RNAiUGP) was rescued by expressing a recoded UGP in the organelle (RNAiUGP/EXPrUGP-GPDH). Our conclusion was supported by targeted metabolomic analyses (IC-HRMS) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones.ImportanceUnusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localisation of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here we showed that the cytosolic and glycosomal pathways are functional. Like in all other eukaryotes, the cytosolic pathways feed glycosylation reactions, however the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK), which are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

Expanding the Enzyme Repertoire for Sugar Nucleotide Epimerization: The CDP-Tyvelose 2-Epimerase from Thermodesulfatator atlanticus for Glucose/Mannose Interconversion

Epimerization of sugar nucleotides is central to the structural diversification of monosaccharide building blocks for cellular biosynthesis. Epimerase applicability to carbohydrate synthesis can be limited, however, by the high degree of substrate specificity exhibited by most sugar nucleotide epimerases. Here, we discovered a promiscuous type of CDP-tyvelose 2-epimerase (TyvE)-like enzyme that promotes C2-epimerization in all nucleotide (CDP, UDP, GDP, ADP, TDP)-activated forms of d-glucose. This new epimerase, originating from Thermodesulfatator atlanticus, is a functional homodimer that contains one tightly bound NAD+/subunit and shows optimum activity at 70°C and pH 9.5. The enzyme exhibits a kcat with CDP-dglucose of ∼1.0 min−1 (pH 7.5, 60°C). To characterize the epimerase kinetically and probe its substrate specificity, we developed chemo-enzymatic syntheses for CDP-dmannose, CDP-6-deoxy-dglucose, CDP-3-deoxy-dglucose and CDP-6-deoxy-dxylo-hexopyranos-4-ulose. Attempts to obtain CDP-dparatose and CDP-dtyvelose were not successful. Using high-resolution carbohydrate analytics and in situ NMR to monitor the enzymatic conversions (60°C, pH 7.5), we show that the CDP-dmannose/CDP-dglucose ratio at equilibrium is 0.67 (± 0.1), determined from the kinetic Haldane relationship and directly from the reaction. We further show that deoxygenation at sugar C6 enhances the enzyme activity 5-fold compared to CDP-dglucose whereas deoxygenation at C3 renders the substrate inactive. Phylogenetic analysis places the T. atlanticus epimerase into a distinct subgroup within the sugar nucleotide epimerase family of SDR (short-chain dehydrogenases/reductases), for which the current study now provides the functional context. Collectively, our results expand an emerging toolbox of epimerase-catalyzed reactions for sugar nucleotide synthesis. IMPORTANCE Epimerases of the sugar nucleotide-modifying class of enzymes have attracted considerable interest in carbohydrate (bio)chemistry, for the mechanistic challenges and the opportunities for synthesis involved in the reactions catalyzed. Discovery of new epimerases with expanded scope of sugar nucleotide substrates used is important to promote the mechanistic inquiry and can facilitate the development of new enzyme applications. Here, a CDP-tyvelose 2-epimerase-like enzyme from Thermodesulfatator atlanticus is shown to catalyze sugar C2 epimerization in CDP-glucose and other nucleotide-activated forms of dglucose. The reactions are new to nature in the context of enzymatic sugar nucleotide modification. The current study explores the substrate scope of the discovered C2-epimerase and, based on modeling, suggests structure-function relationships that may be important for specificity and catalysis.

Targeted Chemoenzymatic Synthesis of Sugar Nucleotide Probes Reveal an Inhibitor of the GDP-D-Mannose Dehydrogenase from Pseudomonas Aeruginosa

<p>Sufferers of the autosomal recessive genetic disorder cystic fibrosis are at extremely high risk for contracting chronic lung infections. Over their lifetime one bacterial strain in particular, <i>Pseudomonas aeruginosa</i>, becomes the dominant pathogen. Bacterial strains incur loss-of-function mutations in the mucA gene that lead to a phenomenon known as mucoid conversion, resulting in copious secretion of alginate, a carbohydrate exopolysaccharide. Strategies that can stop the production of alginate in mucoid <i>Pseudomonas aeruginosa </i>infections are therefore of paramount importance. To aid in this we developed a series of sugar nucleotide chemical tools to probe an enzyme critical to alginate biosynthesis, guanosine diphosphate mannose dehydrogenase (GMD). This enzyme catalyses the irreversible formation of the alginate sugar nucleotide building block, guanosine diphosphate mannuronic acid. Using a chemoenzymatic strategy we accessed a series of modified sugar nucleotides, identifying a C6-amide derivative of the native substrate as a micromolar inhibitor of GMD.<b> </b>This discovery will provide a framework for wider inhibition strategies against GMD to be developed.<b></b></p>

Targeted Chemoenzymatic Synthesis of Sugar Nucleotide Probes Reveal an Inhibitor of the GDP-D-Mannose Dehydrogenase from Pseudomonas Aeruginosa

<p>Sufferers of the autosomal recessive genetic disorder cystic fibrosis are at extremely high risk for contracting chronic lung infections. Over their lifetime one bacterial strain in particular, <i>Pseudomonas aeruginosa</i>, becomes the dominant pathogen. Bacterial strains incur loss-of-function mutations in the mucA gene that lead to a phenomenon known as mucoid conversion, resulting in copious secretion of alginate, a carbohydrate exopolysaccharide. Strategies that can stop the production of alginate in mucoid <i>Pseudomonas aeruginosa </i>infections are therefore of paramount importance. To aid in this we developed a series of sugar nucleotide chemical tools to probe an enzyme critical to alginate biosynthesis, guanosine diphosphate mannose dehydrogenase (GMD). This enzyme catalyses the irreversible formation of the alginate sugar nucleotide building block, guanosine diphosphate mannuronic acid. Using a chemoenzymatic strategy we accessed a series of modified sugar nucleotides, identifying a C6-amide derivative of the native substrate as a micromolar inhibitor of GMD.<b> </b>This discovery will provide a framework for wider inhibition strategies against GMD to be developed.<b></b></p>