Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci.

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 (kcat/Km) 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.

ADPglucose pyrophosphorylase's N-terminus: structural role in allosteric regulation.

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.

Aspartate residue 142 is important for catalysis by ADP-glucose pyrophosphorylase from Escherichia coli.

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.

Activation of the potato tuber ADP-glucose pyrophosphorylase by thioredoxin.

The potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase (ADP-GlcPPase) catalyzes the first committed step in starch biosynthesis. The main type of regulation of this enzyme is allosteric, and its activity is controlled by the ratio of activator, 3-phosphoglycerate to inhibitor, P(i). It was reported (Fu, Y., Ballicora, M. A., Leykam, J. F., and Preiss, J. (1998) J. Biol. Chem. 273, 25045-25052) that the enzyme was activated by reduction of the Cys(12) disulfide linkage present in the catalytic subunits. In this study, both reduced thioredoxin f and m from spinach (Spinacia oleracea) leaves reduced and activated the enzyme at low concentrations (10 microM) of activator (3-phosphoglycerate). Fifty percent activation was at 4.5 and 8.7 microM for reduced thioredoxin f and m, respectively, and 2 orders of magnitude lower than for dithiothreitol. The activation was reversed by oxidized thioredoxin. Cys(12) is conserved in the ADP-GlcPPases from plant leaves and other tissues except for the monocot endosperm enzymes. We postulate that in photosynthetic tissues, reduction could play a role in the fine regulation of the ADP-GlcPPase mediated by the ferredoxin-thioredoxin system. This is the first time that a covalent mechanism of regulation is postulated in the synthesis of starch.

Heat stability of the potato tuber ADP-glucose pyrophosphorylase: role of Cys residue 12 in the small subunit.

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.

Mechanism of reductive activation of potato tuber ADP-glucose pyrophosphorylase.

The potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase activity is activated by a incubation with ADP-glucose and dithiothreitol or by ATP, glucose- 1-phosphate, Ca2+, and dithiothreitol. The activation was accompanied by the appearance of new sulfhydryl groups as determined with 5, 5'-dithiobis(2-nitrobenzoic acid). By analyzing the activated and nonactivated enzymes on SDS-polyacrylamide gel electrophoresis under nonreducing conditions, it was found that an intermolecular disulfide bridge between the small subunits of the potato tuber enzyme was reduced during the activation. Further experiments showed that the activation was mediated via a slow reduction and subsequent rapid conformational change induced by ADP-glucose. The activation process could be reversed by oxidation with 5, 5'-dithiobis(2-nitrobenzoic acid). Incubation with ADP-glucose and dithiothreitol could reactivate the oxidized enzyme. Chemical modification experiments with [14C]iodoacetic acid and 4-vinylpyridine determined that the intermolecular disulfide bridge was located between Cys12 of the small subunits of the potato tuber enzyme. Mutation of Cys12 in the small subunit into either Ala or Ser eliminated the requirement of DTT on the activation and prevented the formation of the intermolecular disulfide of the potato tuber enzyme. The mutants had instantaneous activation rates as the wild-type in the reduced state. A two-step activation model is proposed.

ADP-Glucose pyrophosphorylase from potato tubers. Site-directed mutagenesis studies of the regulatory sites.

Several lysines (Lys) were determined to be involved in the regulation of the ADP-glucose (Glc) pyrophosphorylase from spinach leaf and the cyanobacterium Anabaena sp. PCC 7120 (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706-24711; Y. Charng, A.A. Iglesias, J. Preiss [1994] J Biol Chem 269: 24107-24113). Site-directed mutagenesis was used to investigate the relative roles of the conserved Lys in the heterotetrameric enzyme from potato (Solanum tuberosum L.) tubers. Mutations to alanine of Lys-404 and Lys-441 on the small subunit decreased the apparent affinity for the activator, 3-phosphoglycerate, by 3090- and 54-fold, respectively. The apparent affinity for the inhibitor, phosphate, decreased greater than 400-fold. Mutation of Lys-441 to glutamic acid showed even larger effects. When Lys-417 and Lys-455 on the large subunit were mutated to alanine, the phosphate inhibition was not altered and the apparent affinity for the activator decreased only 9- and 3-fold, respectively. Mutations of these residues to glutamic acid only decreased the affinity for the activator 12- and 5-fold, respectively. No significant changes were observed on other kinetic constants for the substrates ADP-Glc, pyrophosphate, and Mg2+. These data indicate that Lys-404 and Lys-441 on the small subunit are more important for the regulation of the ADP-Glc pyrophosphorylase than their homologous residues in the large subunit.

The reductive modulation of chloroplast fructose-1,6-bisphosphatase by tributylphosphine and sodium borohydride.

The cleavage of disulfide bonds is the major modification of chloroplast fructose-1,6-bisphosphatase when the light-mediated ferredoxin-thioredoxin system enhances the activity of the enzyme. In vitro, only thiol-bearing compounds are functional in the stimulation of fructose 1,6-bisphosphate hydrolysis. This investigation was undertaken to determine the effectivity of other reductants for enhancing the catalytic capacity. In the presence of 1 mM fructose 1,6-bisphosphate and 0.1 mM Ca2+, the five-fold activation triggered by 3.5 mM tributylphosphine is further potentiated by 15% (v/v) 2-propanol. When the enzyme is incubated in the presence of 0.15 M sodium trichloroacetate in place of the cosolvent, NaH4B initially stimulates the activity but subsequently causes the inactivation of the enzyme. A model developed to analyze this dual effect suggests that the concerted action of fructose 1,6-bisphosphate, Ca2+ and trichloroacetate yields an enzyme form that is slightly activable by reduction (t0.5 = 28 min.). However, chloroplast fructose-1,6-bisphosphatase becomes highly sensitive to trichloroacetate inactivation (t0.5 = 5 min.) when NaH4B reduces fructose 1,6-bisphosphate. Hence, the thiol/disulfide exchange constitutes a particular case of reductive mechanisms that stimulate the activity of chloroplast fructose-1,6-bisphosphatase.

Chloroplast fructose-1,6-bisphosphatase: modification of non-covalent interactions promote the activation by chimeric Escherichia coli thioredoxins.

Although all thioredoxins contain a highly conserved amino acid sequence responsible for thiol/disulfide exchanges, only chloroplast thioredoxin-f is effective in the reductive stimulation of chloroplast fructose-1,6-bisphosphatase. We set out to determine whether Escherichia coli thioredoxin becomes functional when selected modulators alter the conformation of the target enzyme. Wild type and chimeric Escherichia coli thioredoxins match the chloroplast counterpart when the activation of chloroplast fructose 1,6-biphosphatase is performed in the presence of fructose 1,6-bisphosphate, Ca2+, and either trichloroacetate or 2-propanol. These modulators of enzyme activity do change the conformation of chloroplast fructose-1,6-bisphosphatase whereas bacterial thioredoxins remain unaltered. Given that fructose 1,6-bisphosphate, Ca2+, and non-physiological perturbants modify non-covalent interactions of the protein but do not participate in redox reactions, these results strongly suggest that the conformation of the target enzyme regulates the rate of thiol/disulfide exchanges catalyzed by protein disulfide oxidoreductases.

Adenosine 5'-diphosphate-glucose pyrophosphorylase from potato tuber. Significance of the N terminus of the small subunit for catalytic properties and heat stability.

cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5'-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081-1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5'-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunit's major function is catalysis.

Enhancement of the reductive activation of chloroplast fructose-1,6-bisphosphatase by modulators and protein perturbants.

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 for photosynthetic CO2 assimilation: enzyme modulation.

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.