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Starch branching enzyme is a highly conserved protein from plants to algae. This enzyme participates in starch granule assembly by the addition of α-1,6-glucan branches to the α-1,4-polyglucans. This modification determines the structure of amylopectin thus arranging the final composition of the starch granule. Herein, we describe the function of the Ot01g03030 gene from the picoalgae Ostreococcus tauri. Although in silico analysis suggested that this gene codes for a starch debranching enzyme, our biochemical studies support that this gene encodes a branching enzyme (BE). The resulting 1058 amino acids protein has two in tandem carbohydrate binding domains (CBMs, from the CBM41 and CBM48 families) at the N-terminal (residues 64-403) followed by the C-terminal catalytic domain (residues 426-1058). Analysis of the BE truncated isoforms show that the CBMs bind differentially to whole starch, amylose or amylopectin. Furthermore, both CBMs seem to be essential for BE activity, as no catalytic activity was detected in the truncated enzyme comprising only by the catalytic domain. Our results suggest that the Ot01g03030 gene codifies for a functional BE containing two CBMs from CBM41 and CBM48 families which are critical for enzyme function and regulation., (Copyright © 2017. Published by Elsevier Inc.)

The effect of temperature on the activity and stability of ADPglucose pyrophosphorylase from Anabaena PCC 7120 was studied. Experimental optima temperatures were found around 37-40 degrees C or 42-45 degrees C, depending on the absence or the presence of allosteric effectors in the assay medium, respectively. In the range of temperature where the enzyme is stable, curved Arrhenius plots were obtained, indicating a transition temperature between 9 and 12 degrees C. Since these results were observed for both the forward and reverse reaction, with two different sets of substrates and two entirely different assay procedures, it seems unlikely that the effect can be on any component of the system other than the enzyme itself. Results suggest that cyanobacterial ADPglucose pyrophosphorylase undergoes conformational changes at different temperatures, rendering structures with different catalytic efficiencies. The different structures of the enzyme were visualized by emission fluorescence. ADPglucose pyrophosphorylase was irreversibly inactivated when exposed to temperatures above 40 degrees C. Inactivation was dependent on temperature and followed first order kinetics. The substrate, ATP, and the allosteric effectors, 3PGA and Pi, effectively protected the enzyme against thermal inactivation. Protection afforded by ATP was affected by MgCl2. These results suggest that the binding of the effectors to the enzyme resulted in conformational changes of the protein, rendering structures more stable to temperature treatments. Similar structures could be adopted by the enzyme in different environments, since the higher stability was observed in media containing either high ionic strength or high hydrophobicity.

The gene encoding ADPglucose synthetase (EC 2.7.7.27) from Agrobacterium tumefaciens was isolated and expressed in Escherichia coli. The recombinant protein was purified to electrophoretic homogeneity in steps including ion-exchange and hydrophobic chromatography. The same purification procedure was utilized to purify ADPglucose synthetase from A. tumefaciens cells. The enzymes from the two sources were purified and characterized and were found to have identical kinetic, regulatory, and structural properties. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only one polypeptide band of 50 kDa was detected. In immunoblotting following electrophoresis, the 50-kDa band reacted with antibodies raised against the Escherichia coli ADPglucose synthetase; there was no reaction with antibodies raised against the spinach enzyme. The immunoreactivity of the A. tumefaciens ADPglucose synthetase was confirmed in antibody neutralization assays. Using gel filtration, the native enzyme was shown to be a tetramer. Fructose 6-phosphate and pyruvate were the most effective activators of the enzyme; maximal activation was observed in the ADPglucose synthesis direction, in which the enzyme was activated about ninefold by fructose 6-phosphate and fivefold by pyruvate. Both activators increased the affinity of the enzyme for the substrates ATP and glucose 1-phosphate. Inorganic orthophospate, ADP, AMP, and pyridoxal phosphate behaved as inhibitors of the enzyme. The distinctive regulatory properties of the enzyme from A. tumefaciens are compared with those of two enterobacterial enzymes and discussed in the context of their deduced amino acid sequences., (Copyright 1998 Academic Press.)

The irreversible thermal inactivation of the sugarcane leaf NADP(+)-malic enzyme was studied at 50 degrees C and pH 7.0 and 8.0. Depending on the preincubation conditions, thermal inactivation followed mono- or biphasic first-order kinetics. A two-step behavior in the irreversible denaturation process was found when protein concentration was sufficiently low. The protein concentration necessary to obtain monlphasic thermal inactivation kinetics was lower at pH 8.0 than at pH 7.0. The results suggest that biphasic inactivation kinetics are the consequence of the existence of two different oligomeric forms of the enzyme (dimer and tetramer), with the dimer being more stable in regards to thermal inactivation. The effects of the substrate and essential cofactors on the thermostability and equilibrium between the dimeric and tetrameric enzyme forms were also studied. Depending on the pH, NADP+, L-malate, and Mg2+ all had a protective effect on the stability of the dimeric and tetrameric species during thermal treatment. However, these ligands showed different effects on the aggregation state of the enzyme. NADP+ and L-malate induced dissociation, especially at pH 8.0, whereas Mg2+ induced aggregation of the protein. By studying the thermal inactivation kinetics at 50 degrees C and different pH values it was observed that the equilibrium between dimers and tetramers was dramatically affected in the range of pH 7.0-8.0. These results suggest that an amino acid residue(s) in the protein with an apparent pKa value of 7.7 needs to be deprotonated to stabilize aggregation of the enzyme to the tetrameric form.

Bromopyruvate is a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase with respect to phosphoenolpyruvate (Ki: 2.3 mM at pH 8). Relatively low concentrations of this compound completely and irreversibly inactivated the enzyme. The inactivation followed pseudo-first-order kinetics. The haloacid combines first with the carboxylase to give a reversible enzyme-bromopyruvate complex and then alkylates the enzyme. The maximum inactivation rate constant was 0.27 min-1 at pH 7.2 and 30 degrees C and the concentration of bromopyruvate giving half-maximum rate of inactivation was 1.8 mM. The inactivation was prevented by the substrate phosphoenolpyruvate, in the absence or presence of MgCl2, and by the competitive inhibitor P-glycolate. Malate afforded protection at pH 7 but not at pH 8. MgCl2 enhanced the inactivation when it was carried out at pH 7; its effect was mainly due to a decrease in the dissociation constant of the complex between bromopyruvate and the enzyme from 2 to 1.4 mM. This behavior was not observed at pH 8. Analysis of the inactivation at different pH suggests that a group of pKa near 7.5 is important for the binding of the reagent to the carboxylase. Determination of the number of sulfhydryl groups of the native and modified enzyme with [3H]-N-ethylmaleimide suggests that the inactivation correlates with the modification of thiol groups in the enzyme. The substrate prevented the modification of these groups. The results suggest that the alkylating reagent modifies cysteinyl residues at the phosphoenolpyruvate binding site of the carboxylase.

NADP-dependent nonphosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) from spinach leaves has been purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation, molecular sieving on Sephadex G-200, DEAE-cellulose, and 2',5'-ADP-Sepharose affinity chromatography. The purified enzyme exhibited a specific activity of 15 mumol (mg protein)-1 min-1 and was characterized as a homotetramer with a native molecular weight of 195,000. Preincubation of the purified enzyme with NADP+ resulted in an almost twofold increase in enzymatic activity. The rate of activation was slower than the rate of catalysis, indicating that the enzyme has hysteretic properties. This behavior results in a lag phase during activity measurement of the enzyme preincubated without NADP+. Substrate interaction and product inhibition studies suggest a rapid equilibrium random BiBi mechanism for the reaction. Thiol modifying reagents, iodoacetamide and diamide, completely inactivated the purified enzyme. Inactivation by iodoacetamide exhibited pseudo-first-order kinetics with a rate constant of 0.17 min-1. D-Glyceraldehyde 3-phosphate effectively protected the enzyme against inactivation by thiol reagents, suggesting that modification occurred at or near the substrate-binding site. Complete inactivation of the dehydrogenase was correlated with incorporation of 8 mol [1-14C]iodoacetamide/mol enzyme. Total protection afforded by D-glyceraldehyde 3-phosphate against enzyme inactivation by iodoacetamide was correlated with a protection of 4 mol reactive residues/mol enzyme. On the basis of these results it is suggested that one sulfhydryl group per enzyme subunit is essential for catalysis in spinach leaf nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase. A kinetic and molecular mechanism for the reaction is proposed.

Phosphoenolpyruvate carboxylase from maize leaves was inactivated by pyridoxal 5'-phosphate in the dark and in the light. A two-step reversible mechanism is proposed for inactivation in the dark, which involves the formation of a noncovalent complex prior to a Schiff base with amino groups of the enzyme. Spectral analysis of pyridoxal 5'-phosphate-modified phosphoenolpyruvate carboxylase showed absorption maxima at 432 and 327 nm, before and after reduction with NaBH4, respectively, suggesting that epsilon-amino groups of lysine residues are the reactive groups in the enzyme. A correlation between spectral data and the maximal inactivation obtained with several concentrations of inhibitor allowed us to establish that the incorporation of 4 mol of pyridoxal 5'-phosphate per mole of holoenzyme accounts for total inactivation. The absence of modifier bound to phosphoenolpyruvate carboxylase when the modification was carried out in the presence of phosphoenolpyruvate and MgCl2 suggests the existence of an essential lysine residue at the catalytic site of the enzyme. Modification of phosphoenolpyruvate carboxylase in the light under an oxygen atmosphere resulted in an irreversible inactivation, which was completely protected by phosphoenolpyruvate and MgCl2. Spectral analysis of the photomodified enzyme showed an absorption peak of 320 nm, suggesting light-mediated addition of a nucleophilic residue (probably an imidazole group) to the pyridoxal 5'-phosphate-lysine azomethine bond.