Partial Purification, Photoaffinity Labeling, and Properties of Mung Bean UDP-Glucose:Dolicholphosphate Glucosyltransferase

UDP-glucose:dolichylphosphate glucosyltransferase has been purified 734-fold from Triton X-100 solubilized mung bean (Phaseolus aureus) microsomes. The partially purified enzyme has broad pH optima of activity from 6.0 to 7.0 and is maximally stimulated with 10 millimolar MgCl₂. The K_m for UDP-glucose was determined as 27 micromolar, and the K_m for dolichol-P was 2 micromolar. Using the UDP-glucose photoaffinity analog, 5-azido-UDP-glucose, a polypeptide of 39 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels was identified as the catalytic subunit of the enzyme. Photoinsertion into this 39-kilodalton polypeptide with [³²P]5-azido-UDP-glucose was saturable, and was maximally protected with the native substrate UDP-glucose. 5-Azido-UDP-glucose behaves competitively with UDP-glucose in enzyme assays, and upon photolysis inhibits activity in proportion to its concentration. This study represents the first subunit identification of a plant glycosyltransferase involved in the biosynthesis of the lipid-linked oligosaccharides that are precursors of N-linked glycoproteins.     

Enhanced UDP-glucose and UDP-galactose by homologous overexpression of UDP-glucose pyrophosphorylase in Lactobacillus casei

UDP-sugars are widely used as substrates in the synthesis of oligosaccharides catalyzed by glycosyltransferases. In the present work a metabolic engineering strategy aimed to direct the carbon flux towards UDP-glucose and UDP-galactose biosynthesis was successfully applied in Lactobacillus casei. The galU gene coding for UDP-glucose pyrophosphorylase (GalU) enzyme in L. casei BL23 was cloned under control of the inducible nisA promoter and it was shown to be functional by homologous overexpression. Notably, about an 80-fold increase in GalU activity resulted in approximately a 9-fold increase of UDP-glucose and a 4-fold increase of UDP-galactose. This suggested that the endogenous UDP-galactose 4-epimerase (GalE) activity, which inter-converts both UDP-sugars, is not sufficient to maintain the UDP-glucose/UDP-galactose ratio. The L. casei galE gene coding for GalE was cloned downstream of galU and the resulting plasmid was transformed in L. casei. The new recombinant strain showed about a 4-fold increase of GalE activity, however this increment did not affect that ratio, suggesting that GalE has higher affinity for UDP-galactose than for UDP-glucose. The L. casei strains constructed here that accumulate high intracellular levels of UDP-sugars would be adequate hosts for the production of oligosaccharides.     

Characterization of mouse UDP-glucose pyrophosphatase, a Nudix hydrolase encoded by the Nudt14 gene

Recombinant mouse UDP-glucose pyrophosphatase (UGPPase), encoded by the Nudt14 gene, was produced in Escherichia coli and purified close to homogeneity. The enzyme catalyzed the conversion of [β-³²P]UDP-glucose to [³²P]glucose-1-P and UMP, confirming that it hydrolyzed the pyrophosphate of the nucleoside diphosphate sugar to generate glucose-1-P and UMP. The enzyme was also active toward ADP-ribose. Activity is dependent on the presence of Mg²⁺ and was greatest at alkaline pH above 8. Kinetic analysis indicated a K_m of ∼4 mM for UDP-glucose and ∼0.3 mM for ADP-ribose. Based on V_max/K_m values, the enzyme was ∼20-fold more active toward ADP-ribose. UGPPase behaves as a dimer in solution and can be cross-linked to generate a species of M_r 54,000 from a monomer of 30,000 as judged by SDS-PAGE. The dimerization was not affected by the presence of glucose-1-P or UDP-glucose. Using antibodies raised against the recombinant protein, Western analysis indicated that UGPPase was widely expressed in mouse tissues, including skeletal muscle, liver, kidney, heart, lung, fat, heart and pancreas with a lower level in brain. It was generally present as a doublet when analyzed by SDS-PAGE, suggesting the occurrence of some form of post-translational modification. Efforts to interconvert the species by adding or inhibiting phosphatase activity were unsuccessful, leaving the nature of the modification unknown. Sequence alignments and database searches revealed related proteins in species as distant as Drosophila melanogaster and Caenorhabditis elegans.     

Purification and properties of UDP-glucose: coniferyl alcohol glucosyltransferase from suspension culturesof Paul's scarlet rose.

UDP-glucose:coniferyl alcohol glucosyltransferase was isolated from 10-day-old, darkgrown cell suspension cultures of Paul's scarlet rose. The enzyme was purified 120-fold by (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-100. The enzyme has a pH optimum of 7.5 in Tris-HCl buffer, required an -SH group for activity, and is inhibited by ?-chloromercuribenzoate and EDTA. Its molecular weight is estimated to be 52,000. The enzyme is specific for the glucosylation of coniferyl alcohol (K_m 3.3 × 10^?6 M) and sinapyl alcohol (K_m 5.6 × 10^?6 M). With coniferyl alcohol as substrate the apparent K_m value for UDP-glucose is 2 × 10^?6m. The enzyme activity can be detected in a number of callus-tissue and cell-suspension cultures. The role of this enzyme is believed to be to catalyze the transfer of glucose from UDPG to coniferyl (or sinapyl) alcohol as storage intermediates in lignin biosynthesis.     

Pyrophosphorylases in Solanum tuberosum: II. CATALYTIC PROPERTIES AND REGULATION OF ADP-GLUCOSE AND UDP-GLUCOSE PYROPHOSPHORYLASE ACTIVITIES IN POTATOES

Pyrophosphorylytic kinetic constants (S_0.5, V_max) of partially purified UDP-glucose- and ADP-glucose pyrophosphorylases from potato tubers were determined in the presence of various intermediary metabolites. The S_0.5 of UDP-glucose pyrophosphorylase for UDP-glucose (0.17 millimolar) or pyrophosphate (0.30 millimolar) and the V_max were not influenced by high concentrations (2 millimolar) of these substances. The most efficient activator of ADP-glucose pyrophosphorylase was 3-P-glycerate (A_0.5 = 4.5 × 10⁻⁶ molar). The S_0.5 for ADP-glucose and pyrophosphate was increased 3.5-fold (0.83 to 0.24 millimolar) and 1.8-fold (0.18 to 0.10 millimolar), respectively, with 0.1 millimolar 3-P-glycerate while the V_max was increased nearly 4-fold. The magnitude of 3-P-glycerate stimulation was dependent upon the integrity of key sulfhydryl groups (−SH) and pH. Oxidation or blockage of −SH groups resulted in a marked reduction of enzyme activity. Stimulations of 3.1-, 2.9-, 4.8-, and 9.5-fold were observed at pH 7.5, 8.0, 8.5, and 9.0, respectively, in the presence of 3-P-glycerate (2 millimolar). The most potent inhibitor of ADP-glucose pyrophosphorylase was orthophosphate (I_0.5 = 8.8 × 10⁻⁵. molar). This inhibition was reversed with 3-P-glycerate (1.2 × 10⁻⁴ molar), resulting in an increased I_0.5 value of 1.5 × 10⁻³ molar. Likewise, orthophosphate (7.5 × 10⁻⁴ molar) caused a decrease in the activation efficiency of 3-P-glycerate (A_0.5 from 4.5 × 10⁻⁶ molar to 6.7 × 10⁻⁵ molar). The significance of 3-P-glycerate activation and orthophosphate inhibition in the regulation of α-glucan biosynthesis in Solanum tuberosum is discussed.     

Purification of a UTP: d-Glucose-1-phosphate uridylyl-transferase from Golgi apparatus of cat liver by affinity chromatography on UTP-agarose

UDP-glucose pyrophosphorylase from Golgi apparatus solubilized by detergent has been purified 100-fold from microsomes by affinity chromatography on UTP-agarose. The purified enzyme has apparent M_r 270,000 and isoelectric pH 3.9 against 360,000 and 4.2 for soluble enzyme. According to these characteristics, UDP-glucose pyrophosphorylase from Golgi apparatus is different from cytosolic enzyme.     

Trehalose 6-phosphate synthase from Dictyostelium discoideum: partial purification and characterization of the enzyme from young sorocarps.

Trehalose 6-phosphate synthase was solubilized from young sorocarps of the cellular slime mold, Dictyostelium discoideum, by a freeze-thaw cycle and was subsequently purified about 160-fold using streptomycin sulfate precipitation, (NH₄)₂SO₄ fractionation, DEAE-cellulose chromatography, heat treatment in the presence of heparin, and molecular sieve chromatography on columns of Bio-Gel A-1.5m. The purified enzyme was maximally active at pH 6.5, showed an absolute specificity for glucose 6-phosphate as glucosyl acceptor and a relative specificity for the glucosyl donor in the order: UDP-glucose, GDP-glucose, and ADP-glucose. Although heparin and chondroitin sulfate activated the synthase, the order of glucosyl donor specificity was not affected. Other activators of trehalose 6-phosphate synthase were KCL, Mg²⁺, and EDTA, while detergents had little effect. Although synthase activity was reduced 60 to 80% upon the omission of Mg²⁺ from the assay mixture, an absolute dependency for Mg²⁺ could not be demonstrated. Evaluation of the apparent K_m values for partially purified synthase preparations demonstrated that for each of the synthase substrates, the Line weaver-Burk plots displayed complex bimodal kinetics. Estimation of the Michaelis constants after extrapolation of the straight line portions of these plots yielded values of (a) 0.2 and 3.2 mm glucose 6-phosphate and (b) 0.5 and 2.2 mm UDP-glucose. Comparison of the latter parameters with the cellular levels of UDP-glucose and glucose 6-phosphate in Dictyostelium suggests that if the observed bimodal kinetics are the consequence of multiple kinetically distinct forms of the synthase, the activation of trehalose synthesis during slime mold culmination could provide a rationale for the presence of these isozymes.     

Direct Photolabeling with [P]UDP-Glucose for Identification of a Subunit of Cotton Fiber Callose Synthase

We have identified a 52 kilodalton polypeptide as being a likely candidate for the catalytic subunit of the UDP-glucose: (1→3)-β-glucan (callose) synthase of developing fibers of Gossypium hirsutum (cotton). Such a polypeptide migrates coincident with callose synthase during glycerol gradient centrifugation in the presence of EDTA, and can be directly photolabeled with the radioactive substrate, α-[³²P]UDP-glucose. Interaction with the labeled probe requires Ca²⁺, a specific activator of callose synthase which is known to lower the K_m of higher plant callose synthases for the substrate UDP-glucose. Using this probe and several other related ones, several other proteins which interact with UDP-glucose were also identified, but none satisfied all of the above criteria for being components of the callose synthase.     

Functional Analysis of the Lactococcus lactis galU and galE Genes and Their Impact on Sugar Nucleotide and Exopolysaccharide Biosynthesis

We studied the UDP-glucose pyrophosphorylase (galU) and UDP-galactose epimerase (galE) genes of Lactococcus lactis MG1363 to investigate their involvement in biosynthesis of UDP-glucose and UDP-galactose, which are precursors of glucose- and galactose-containing exopolysaccharides (EPS) in L. lactis. The lactococcal galU gene was identified by a PCR approach using degenerate primers and was found by Northern blot analysis to be transcribed in a monocistronic RNA. The L. lactis galU gene could complement an Escherichia coli galU mutant, and overexpression of this gene in L. lactis under control of the inducible nisA promoter resulted in a 20-fold increase in GalU activity. Remarkably, this resulted in approximately eightfold increases in the levels of both UDP-glucose and UDP-galactose. This indicated that the endogenous GalE activity is not limiting and that the GalU activity level in wild-type cells controls the biosynthesis of intracellular UDP-glucose and UDP-galactose. The increased GalU activity did not significantly increase NIZO B40 EPS production. Disruption of the galE gene resulted in poor growth, undetectable intracellular levels of UDP-galactose, and elimination of EPS production in strain NIZO B40 when cells were grown in media with glucose as the sole carbon source. Addition of galactose restored wild-type growth in the galE disruption mutant, while the level of EPS production was approximately one-half the wild-type level.     

Structural basis for the reaction mechanism of UDP-glucose pyrophosphorylase

UDP-glucose pyrophosphorylases (UGPase; EC 2.7.7.9) catalyze the conversion of UTP and glucose-1-phosphate to UDP-glucose and pyrophosphate and vice versa. Prokaryotic UGPases are distinct from their eukaryotic counterparts and are considered appropriate targets for the development of novel antibacterial agents since their product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharides and capsular polysaccharides. In this study, the crystal structures of UGPase from Helicobacter pylori (HpUGPase) were determined in apo- and UDP-glucose/Mg²⁺-bound forms at 2.9 Å and 2.3 Å resolutions, respectively. HpUGPase is a homotetramer and its active site is located in a deep pocket of each subunit. Magnesium ion is coordinated by Asp130, two oxygen atoms of phosphoryl groups, and three water molecules with octahedral geometry. Isothermal titration calorimetry analyses demonstrated that Mg²⁺ ion plays a key role in the enzymatic activity of UGPase by enhancing the binding of UGPase to UTP or UDP-glucose, suggesting that this reaction is catalyzed by an ordered sequential Bi Bi mechanism. Furthermore, the crystal structure explains the specificity for uracil bases. The current structural study combined with functional analyses provides essential information for understanding the reaction mechanism of bacterial UGPases, as well as a platform for the development of novel antibacterial agents.     

UDP-glucose Pyrophosphorylase from Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

UDP-glucose pyrophosphorylase of Jerusalem artichoke tubers was purified 90-fold over the crude extract. The purified enzyme preparation absolutely required magnesium ions for activity. Cobalt ions were 60% as effective as magnesium ions; other divalent cations including manganese showed little or no effect. This enzyme had a pH optimum of 8.5 and a temperature optimum of 40°C. ATP and UDP inhibited the activity of this enzyme in both forward and backward directions. Km values for UDP-glucose, inorganic pyrophosphate, glucose-1-phosphate and UTP were determined to be 4.45 × 10^?4 M, 2.33 × 10^?4 M, 9.38 × 10^?4 M and 2.98 × 10^?4 M, respectively. These results are discussed in comparison with those of UDP-glucose pyrophosphorylases isolated from other plants.     

Analysis of glucose 1-phosphate at the picomole level with [C]UTP and uridine 5′-diphosphoglucose pyrophosphorylase

A new sensitive method is described for glucose 1-phosphate analysis. The key reaction is the pyrophosphorolysis of UDP-glucose catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase. The reaction product, [¹⁴C]UDP-glucose, is separated from [¹⁴C]UTP by adsorbing [¹⁴C]UTP selectively onto polyethyleneimine cellulose or by separating both labeled compounds on one-dimensional polyethyleneimine thin-layer chromatograms. The sensitivity of the method for glucose 1-phosphate analysis is 5 pmol. The method has been successfully employed to monitor the level of glucose 1-phosphate in early germination of wheat embryos.     

UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product

A re-examination of the kinetic properties of UDP-glucose: (1→3)-β-glucan (callose) synthases from mung bean seedlings (Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca²⁺ and millimolar concentrations of β-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V_max of the enzyme, and by lowering the apparent K_m for UDP-glucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg²⁺ markedly enhances the affinity of the mung bean enzyme for Ca²⁺ but not for β-glucoside; with saturating Ca²⁺, Mg²⁺ only slightly stimulates further production of glucan. However, the presence of Mg²⁺ during synthesis, or NaBH₄ treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca²⁺, β-glucoside-activated cotton fiber enzyme, with or without Mg²⁺, is very similar in size to callose isolated from cotton fibers, but is a linear (1→3)-β-glucan lacking the small amount of branches at C-0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg²⁺in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg²⁺ on the conformation of the enzyme. Rate-zonal centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.     

Pyrophosphorylases in Solanum tuberosum: I. Changes in ADP-Glucose and UDP-Glucose Pyrophosphorylase Activities Associated with Starch Biosynthesis during Tuberization, Maturation, and Storage of Potatoes

Changes in ADP-glucose and UDP-glucose pyrophosphorylase activities were followed during tuber development of Solanum tuberosum and prolonged storage at 4 and 11 C. Potato tuberization was accompanied by a sharp increase in starch synthesis simultaneous with a marked rise in ADP-glucose pyrophosphorylase activity. When tubers reached an average diameter of 1 centimeter (0.5 gram average tuber weight) and had already established 58% starch on a dry weight basis, ADP-glucose pyrophosphorylase increased 16- to 24-fold over its activity seen in low starch containing stolon tissue. During this same period UDP-glucose pyrophosphorylase increased approximately 2- to 3-fold. Although participation of UDP-glucose in starch formation can not be neglected, it is suggested that the onset of rapid non-photosynthetic potato tuber starch biosynthesis may be closely related to the simultaneous increase in ADP-glucose pyrophosphorylase activity.     

Labeling of the Plasma Membrane of Pea Cells by a Surface-localized Glucan Synthetase

When radioactive UDP-glucose is supplied to 1-millimeter-thick slices of pea (Pisum sativum) stem tissue, radioactive glucose becomes incorporated into membrane-bound polysaccharides. Evidence is given that this incorporation does not result from breakdown of UDP-glucose and utilization of the resultant free glucose, and that the incorporation most likely takes place at the cell surface, leading to a specific labeling of the plasma membrane. The properties of the plasma membrane that are indicated by this method of recognition, including the association of K⁺-stimulated ATPase activity with the plasma membrane, resemble properties inferred using other approaches. The membrane-associated polysaccharide product formed from UDP-glucose is largely 1,3-linked glucan, presumably callose, and does not behave as a precursor of cell wall polymers. No substantial amount of cellulose is formed from UDP-glucose in this procedure, even though these cells incorporate free glucose rapidly into cellulose. This synthetase system that uses external UDP-glucose may serve for formation of wound callose.     

Formation of UDP-Xylose and Xyloglucan in Soybean Golgi Membranes

Soybean (Glycine max) membranes co-equilibrating with Golgi vesicles in linear sucrose gradients contained UDP-glucuronate carboxy-lyase and xyloglucan synthase activities. Digitonin solubilized and increased the activity of the membrane-bound UDP-glucuronate carboxy-lyase. UDP-xylose did not inhibit the transport of UDP-glucuronate into the lumen of Golgi vesicles but repressed the decarboxylation of the translocated UDP-glucuronate. The results suggest that UDP-glucuronate is transported into the vesicles by a specific carrier and decarboxylated to UDP-xylose within the lumen. On incubation of UDP-[¹⁴C]glucuronate with Golgi membranes in the presence of UDP-glucose, [¹⁴C]xylose-labeled xyloglucan was formed. Although the K_m value of UDP-glucuronate for the decarboxylation was 240 micromolar, the affinity of UDP-glucuronate for xyloglucan formation (31 micromolar) was similar to that of UDP-xylose (28 micromolar), suggesting a high turnover of UDP-xylose. The biosynthesis of UDP-xylose from UDP-glucuronate probably occurs in Golgi membranes, where xyloglucan subsequently forms from UDP-xylose and UDP-glucose.     

Purification and Properties of UDP-GlcNAc:Dolichyl-Pyrophosphoryl-GlcNAc GlcNAc Transferase from Mung Bean Seedling

The N-acetylglucosamine (GlcNAc) transferase that catalyzes the formation of dolichyl-pyrophosphoryl-GlcNAc-GlcNAc from UDP-GlcNAc and dolichyl-pyrophosphoryl-GlcNAc was solubilized from the microsomal enzyme fraction of mung beans with 1.5% Triton X-100, and was purified 140-fold on columns of DE-52 and hydroxylapatite. The partially purified enzyme preparation was quite stable when stored in 20% glycerol and 0.5 millimolar dithiothreitol, and was free of GlcNAc-1-P transferase and mannosyl transferases. The GlcNAc transferase had a sharp pH optimum of 7.4 to 7.6 and the K_m for dolichyl-pyrophosphoryl-GlcNAc was 2.2 micromolar and that for UDP-GlcNAc, 0.25 micromolar. The enzyme showed a strong requirement for the detergent Triton X-100 and was stimulated somewhat by the divalent cation Mg²⁺. Uridine nucleotides, especially UDP and UDP-glucose inhibited the enzyme as did the antibiotic, diumycin. However, a variety of other antibiotics including tunicamycin were without effect. The product of the reaction was characterized as dolichyl-pyrophosphoryl-GlcNAc-GlcNAc.     

UDP-galactose 4′-epimerase from vicia faba seeds

UDP-Galactose 4′-epimerase was purified ca 800-fold through a multi-step procedure which included affinity chromatography using NAD⁺ -Agarose. Three forms of the enzyme were separated by gel-filtration but only the major form was purified. The pH optimum of the enzyme was 9.5. Exogenous NAD⁺ was not required for enzymic activity but its removal caused inactivation. The enzyme was unstable below pH 7.0 but stable at pH 8.0 in the presence of glycerol and at ?20° for two months. The equilibrium constant for the enzyme-catalysed reaction was 3.2 ± 0.15. The K_m for UDP-galactose and UDP-glucose were 0.12 mM and 0.25 mM, respectively. The inhibition by NADH was competitive, with a K_i of 5 μM. The MW of the enzyme was 78 000; the two minor forms showed the values of 158 000 and 39 000, respectively.     

Regulation of glucose metabolism and cell wall synthesis in Avena stem segments by gibberellic Acid

Gibberellic acid (GA) stimulated both the elongation of Avena sativa stem segments and increased synthesis of cell wall material. The effects of GA on glucose metabolism, as related to cell wall synthesis, have been investigated in order to find specific events regulated by GA. GA caused a decline in the levels of glucose, glucose 6-phosphate, and fructose 6-phosphate if exogenous sugar was not supplied to the segments, whereas the hormone caused no change in the levels of glucose 6-phosphate, fructose 6-phosphate, UDP-glucose, or the adenylate energy charge if the segments were incubated in 0.1 m glucose. No GA-induced change could be demonstrated in the activities of hexokinase, phosphoglucomutase, UDP-glucose pyrophosphorylase, or polysaccharide synthetases using UDP-glucose, UDP-galactose, UDP-xylose, and UDP-arabinose as substrates. GA stimulated the activity of GDP-glucose-dependent β-glucan synthetase by 2- to 4-fold over the control. When glucan synthetase was assayed using UDP-glucose as substrate, only β-1,3-linked glucan was synthesized in vitro, whereas with GDP-glucose, only β-1,4-linked glucan was synthesized. These results suggest that one part of the mechanism by which GA stimulates cell wall synthesis concurrently with elongation in Avena stem segments may be through a stimulation of cell wall polysaccharide synthetase activity.     

Rossmann-fold motifs can confer multiple functions to metabolic enzymes: RNA binding and ribonuclease activity of a UDP-glucose dehydrogenase

Metabolic enzymes are usually characterized to have one specific function, and this is the case of UDP-glucose dehydrogenase that catalyzes the twofold NAD⁺-dependent oxidation of UDP-glucose into UDP-glucuronic acid. We have determined that this enzyme is also capable of participating in other cellular processes. Here, we report that the bacterial UDP-glucose dehydrogenase (UgdG) from Sphingomonas elodea ATCC 31461, which provides UDP-glucuronic acid for the synthesis of the exopolysaccharide gellan, is not only able to bind RNA but also acts as a ribonuclease. The ribonucleolytic activity occurs independently of the presence of NAD⁺ and the RNA binding site does not coincide with the NAD⁺ binding region. We have also performed the kinetics of interaction between UgdG and RNA. Moreover, computer analysis reveals that the N- and C-terminal domains of UgdG share structural features with ancient mitochondrial ribonucleases named MAR. MARs are present in lower eukaryotic microorganisms, have a Rossmannoid-fold and belong to the isochorismatase superfamily. This observation reinforces that the Rossmann structural motifs found in NAD⁺-dependent dehydrogenases can have a dual function working as a nucleotide cofactor binding domain and as a ribonuclease.