Search

Abstract Background ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the synthesis of glycogen in bacteria and starch in algae and plants. In oxygenic photosynthetic organisms, ADP-Glc PPase is mainly activated by 3-phosphoglycerate (3-PGA) and to a lesser extent by other metabolites. In this work, we analyzed the activation promiscuity of ADP-Glc PPase subunits from the cyanobacterium Anabaena PCC 7120, the green alga Ostreococcus tauri, and potato (Solanum tuberosum) tuber by comparing a specificity constant for 3-PGA, fructose-1,6-bisphosphate (FBP), fructose-6-phosphate, and glucose-6-phosphate. Results The 3-PGA specificity constant for the enzymes from Anabaena (homotetramer), O. tauri, and potato tuber was considerably higher than for other activators. O. tauri and potato tuber enzymes were heterotetramers comprising homologous small and large subunits. Conversely, the O. tauri small subunit (OtaS) homotetramer was more promiscuous because its FBP specificity constant was similar to that for 3-PGA. To explore the role of both OtaS and OtaL (O. tauri large subunit) in determining the specificity of the heterotetramer, we knocked out the catalytic activity of each subunit individually by site-directed mutagenesis. Interestingly, the mutants OtaSD148A/OtaL and OtaS/OtaLD171A had higher specificity constants for 3-PGA than for FBP. Conclusions After gene duplication, OtaS seemed to have lost specificity for 3-PGA compared to FBP. This was physiologically and evolutionarily feasible because co-expression of both subunits restored the specificity for 3-PGA of the resulting heterotetrameric wild type enzyme. This widespread promiscuity seems to be ancestral and intrinsic to the enzyme family. Its presence could constitute an efficient evolutionary mechanism to accommodate the ADP-Glc PPase regulation to different metabolic needs.

Growing antibiotic resistance by pathogenic bacteria has led to a global crisis. The bacterial enzyme N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) provides a very attractive target for the discovery of a new class of antibiotics, as it resides exclusively in many pathogenic bacterial strains and is a key enzyme in the lysine biosynthetic pathway. This pathway is responsible for the production of lysine as well as meso-diaminopimelate (m-DAP), both of which are required for peptidoglycan cell-wall synthesis, and lysine for peptide synthesis. The enzyme DapE catalyzes the hydrolysis of N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) to succinate and l,l-diaminopimelic acid (l,l-DAP), and due to its absence in humans, inhibition of DapE avoids mechanism-based side effects. We have executed the asymmetric synthesis of N,N-dimethyl-SDAP, an l,l-SDAP substrate analog and an analog of the synthetic substrate of our previously described DapE assay. Previous modeling studies advocated that N,N-dimethyl-SDAP might function as an inhibitor, however the compound behaves as a substrate, and we have demonstrated the use of N,N-dimethyl-SDAP as the substrate in a modified ninhydrin-based DapE assay. Thermal shift experiments of DapE in the presence of N,N-dimethyl-SDAP are consistent with a melt temperature (T m ) shifted by succinate, the product of enzymatic hydrolysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Backgound: Studying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy., Methods: We report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (k cat /K m ) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2., Results: All enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules., Conclusions: Results support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications., General Significance: Results presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We studied the functional role of the Escherichia coli ADPglucose pyrophosphorylase's N-terminus in allosteric regulation, and the particular effects caused by its length. Small truncated mutants were designed, and those lacking up to 15-residues were active and highly purified for further kinetic analyses. Ndelta3 and Ndelta7 did not change the kinetic parameters with respect to the wild-type. Ndelta11 and Ndelta15 enzymes were insensitive to allosteric regulation and highly active in the absence of the activator. Co-expression of two polypeptides corresponding to the N- and C-termini generated an enzyme with activation properties lower than those of the wild-type [C.M. Bejar, M.A. Ballicora, D.F. Gómez Casati, A.A. Iglesias, J. Preiss, The ADPglucose pyrophosphorylase from Escherichia coli comprises two tightly bound distinct domains, FEBS Lett. 573 (2004) 99-104]. Here, we characterized a Ndelta15 co-expression mutant, in which the allosteric regulation was restored to wild-type levels. Unusual allosteric effects caused by either an N-terminal truncation or co-expression of individual domains may respond to structural changes favoring an up-regulated or a down-regulated conformation rather than specific activator or inhibitor sites' disruption.

Structural prediction of several bacterial and plant ADP-glucose pyrophosphorylases, as well as of other sugar-nucleotide pyrophosphorylases, was used for comparison with the three-dimensional structures of two crystallized pyrophosphorylases (Brown, K., Pompeo, F., Dixon, S., Mengin-Lecreulx, D., Cambillau, C., and Bourne, Y. (1999) EMBO J. 18, 4096-4107; Blankenfeldt, W., Asuncion, M., Lam, J. S., and Naismith, J. H. (2000) EMBO J. 19, 6652-6663). This comparison led to the discovery of highly conserved residues throughout the superfamily of pyrophosphorylases despite the low overall homology. One of those residues, Asp(142) in the ADP-glucose pyrophosphorylase from Escherichia coli, was predicted to be near the substrate site. To elucidate the function that Asp(142) might play in the E. coli ADP-glucose pyrophosphorylase, aspartate was replaced by alanine, asparagine, or glutamate using site-directed mutagenesis. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of the purified mutants showed a decrease in specific activity of up to 4 orders of magnitude. Comparison of other kinetic parameters, i.e. the apparent affinities for substrates and allosteric effectors, showed no significant changes, excluding this residue from the specific role of ligand binding. Only the D142E mutant exhibited altered K(m) values but none as pronounced as the decrease in specific activity. These results show that residue Asp(142) is important in the catalysis of the ADP-glucose pyrophosphorylase from E. coli.

Most of the ADP-glucose pyrophosphorylases from different sources are stable to a heat treatment. We found that in the potato (Solanum tuberosum L.) tuber enzyme, the intermolecular disulfide bridge located between Cys12 of the small subunits is responsible for the stability at 60 degrees C. When this unique disulfide bond is cleaved the enzyme is stable up to 40 degrees C. Mutation of Cys12 in the small subunit into either Ala or Ser yielded enzymes with stability similar to the reduced form of the wild type. Concurrently, the enzyme with a truncated small subunit on the N-terminal was stable only up to 40 degrees C. Thus, the N-terminal is important for the stability of the enzyme because of the presence of a disulfide bond., (Copyright 1999 Academic Press.)

To characterize the mechanism of chloroplast fructose-1,6-bisphosphatase activation, we have examined kinetic and structural changes elicited by protein perturbants and reductants. At variance with its well-known capacity for enzyme inactivation, 150 mM sodium trichloroacetate yielded an activatable chloroplast fructose-1,6-bisphosphatase in the presence of 1.0 mM fructose 1,6-bisphosphate and 0.1 mM Ca2+. Other sugar bisphosphates did not replace fructose 1,6-bisphosphate whereas Mg2+ and Mn2+ were functional in place of Ca2+. Variations of the emission fluorescence of intrinsic fluorophores and a noncovalently bound extrinsic probe [2-(p-toluidinyl)naphthalene-6-sulfonate] indicated the presence of conformations different from the native form. A similar conclusion was drawn from the analysis of absorption spectra by means of fourth-derivative spectrophotometry. The effect of these conformational changes on the reductive process was studied by subsequently incubating the enzyme with dithiothreitol. The reaction of chloroplast fructose-1,6-bisphosphatase with dithiothreitol was accelerated 13-fold by the chaotropic anion: second-order rate constants were 48.1 M-1.min-1 and 3.7 M-1.min-1 in the presence and in the absence of trichloroacetate, respectively. Thus, the enhancement of the reductive activation by compounds devoid of redox activity illustrated that the modification of intramolecular noncovalent interactions of chloroplast fructose-1,6-bisphosphatase plays an essential role in the conversion of enzyme disulfide bonds to sulfhydryl groups. In consequence, a conformational change would operate concertedly with the reduction of disulfide bridges in the light-dependent activation mediated by the ferredoxin-thioredoxin system.

The reductive pentose phosphate cycle (Benson-Calvin cycle) is the main biochemical pathway for the conversion of atmospheric CO2 to organic compounds. Two unique systems that link light-triggered events in thylakoid membranes with enzyme regulation are located in the soluble portion of chloroplasts (stroma): the ferredoxin-thioredoxin system and ribulose 1,5-bisphosphate carboxylase/oxygenase-Activase (Rubisco-Activase). The ferredoxin-thioredoxin system (ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin) transforms native (inactive) glyceraldehyde-3-P dehydrogenase, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, and phosphoribulokinase to catalytically competent forms. However, the comparison of enzymes reveals the absence of common amino acid sequences for the action of reduced thioredoxin. Thiol/disulfide exchanges appear as the underlying mechanism, but chloroplast metabolites and target domains make the activation process peculiar for each enzyme. On the other hand, Rubisco-Activase facilitates the combination of CO2 with a specific epsilon-amino group of ribulose 1,5-bisphosphate carboxylase/oxygenase and the subsequent stabilization of the carbamylated enzyme by Mg2+, in a reaction that depends on ATP and ribulose 1,5-bisphosphate. Most of these studies were carried out in homogeneous solutions; nevertheless, a growing body of evidence indicates that several enzymes of the cycle associate either with thylakoid membranes or with other proteins yielding supra-molecular complexes in the chloroplast.