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A substantial number of plant genes have been identified and isolated recently, and methods for the rapid identification of other genes that are difficult to isolate using present techniques have been devised. Alterations to fundamental aspects of plant growth, and also to the structure and amount of storage products, are now possible. A wide variety of crop plants can now be transformed and a rapid increase in our knowledge of gene regulation in plants is taking place. The manipulation of plant genes for the benefit of argriculture now looks more promising than ever before.

ADPGlucose Pyrophosphorylase (ADPG PPase, glgC gene product) catalyzes a key step in glucan synthesis and is allosterically regulated. Engineering of the ADPG PPase from Rhodopseudomonas palustris ...

ADPglucose pyrophosphorylase catalyzes the regulatory step in the pathway for bacterial glycogen synthesis. The enzymes from different organisms exhibit distinctive regulatory properties related to the main carbon metabolic pathway. Escherichia coli ADPglucose pyrophosphorylase is mainly activated by fructose 1,6-bisphosphate (FBP), whereas the Agrobacterium tumefaciens enzyme is activated by fructose 6-phosphate (F6P) and pyruvate. Little is known about the regions determining the specificity for the allosteric regulator. To study the function of different domains, two chimeric enzymes were constructed. "AE" contains the N-terminus (271 amino acids) of the A. tumefaciens ADPglucose pyrophosphorylase and the C-terminus (153 residues) of the E. coli enzyme, and "EA", the inverse construction. Expression of the recombinant wild-type and chimeric enzymes was performed using derivatives of the pET24a plasmid. Characterization of the purified chimeric enzymes showed that the C-terminus of the E. coli enzyme is relevant for the selectivity by FBP. However, this region seems to be less important for the specificity by F6P in the A. tumefaciens enzyme. The chimeric enzyme AE is activated by both FBP and F6P, neither of which affect EA. Pyruvate activates EA with higher apparent affinity than AE, suggesting that the C-terminus of the A. tumefaciens enzyme plays a role in the binding of this effector. The allosteric inhibitor site is apparently disrupted, as a marked desensitization toward AMP was observed in the chimeric enzymes. Fil: Ballicora, Miguel A.. Michigan State University; Estados Unidos Fil: Sesma, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Preiss, Jack. Michigan State University; Estados Unidos

ADP-glucose pyrophosphorylase (AGPase), a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA) method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies) of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield., Author Summary ADP-glucose pyrophosphorylase (AGPase) is a key heterotetrameric allosteric enzyme involved in plant starch biosynthesis. In this study, we have applied computational and experimental methods to identify critical amino acids of the AGPase large and small subunits that interact with each other during the heterotetrameric structure formation. During the comparison of the computational with the experimental results we also noted that the backbone energy contribution of the interface residues is more important in identifying critical residues. This study will enable us to use a rational approach to obtain better assembled mutant AGPase variants and use them for the improvement of the plant yield.

Grain filling in Maize is mainly dependent on photosynthetic carbon fixed after pollination(l). However in spite of the high sink demand, photosynthetic rate tends to decline because of leaf senescence(2). This phenomenon is probably linked to N remobilization needed for grain protein synthesis.

ADPglucose pyrophosphorylase from the Crassulacean acid metabolism plants Hoya carnosa and Xerosicyos danguyi were partially purified to study their regulatory and kinetic properties. The molecular weight of the native enzymes from both plants was determined to be about 209,000. The enzyme from both plants was found to be activated by glycerate 3-phosphate and inhibited by inorganic phosphate. The kinetic constants for the substrates and Mg2+ are reported. The significance of the activation by glycerate 3-phosphate and inhibition by inorganic phosphate of ADPglucose synthesis catalyzed by the H. carnosa and X, danguyi enzymes is discussed. ADPglucose synthesized by the above enzymes was found to be the most effective donor of the glucosyl portion to α-glucan primer in the starch synthase reaction observed in CAM plants.

ADPglucose pyrophosphorylase level and mechanisms regulating its activity were studied in cucumber plants infected with the cucumber mosaic virus at the stage of chronic infection. Studies carried out with partially purified preparations of the enzyme have shown that there was no substantial difference in the regulatory influence of the ratio 3-PGA/P1, or in the number of binding sites of the effectors on the enzyme, but that the virus infection reduced the level of the enzyme in the tissues to 74% of the control and the 3-PGA/P1 ratio to one half which resulted in a further decrease in ADPglucose pyrophosphorylase activity. In crude homogenate prepared from diseased plants, activity of the enzyme was reduced to 42% of the healthy control.

ADPglucose pyrophosphorylase fromRhodospirillum rubrum has been purified to homogeneity or near homogeneity using affinity chromatography techniques. The subunit molecular weight of the enzyme is 50,000. Thus, the enzyme is similar in subunit molecular weight to that found for other bacterial ADPglucose pyrophosphorylases. The amino acid composition is similar to that found for theRhodospirillum tenue enzyme. However, the N-terminal amino acid sequence of theR. rubrum enzyme shows no apparent homology with theR. tenue enzyme N-terminal amino acid sequence.

Etude du metabolisme des hydrates de carbone dans l'albumen de grains de riz (Oryza sativa) en developpement par comparaison des taux d'activite de 33 enzymes majeures entre l'albumen et les feuilles vertes. Les taux de metabolites intermediaires et la localisation des enzymes cle du metabolisme du saccharose et de l'amidon ont ete etudies dans des amyloplastes de grains de riz en developpement, et ont permis de caracteriser la voie de synthese de l'amidon a partir du saccharose

A new, rapid and sensitive assay of ADPglucose pyrophosphorylase using the luciferaseluciferin system to quantify the reaction product, ATP, is described. The total assay time per sample is about 10 min. The sensitivity ranges from picomoles to nanomoles, and the crude enzyme extract from indicidual seeds of cereals can be assayed.

The ADPglucose pyrophosphorylases of 7 plant-leaf tissues were partially purified and characterized. In all cases the enzymes showed stability to heat treatment at 65° for 5 minutes in the presence of 0.02 m phosphate buffer, pH 7.0. The leaf ADPglucose pyrophosphorylases were activated 5 to 15-fold by 3-phosphoglycerate. Fructose-6-phosphate and fructose 1, 6-diphosphate stimulated ADPglucose pyrophosphorylase to lesser extents. The A 0.5 (conc of activator required to give 50% of the observed maximal activation) of 3-phosphoglycerate for the barley enzyme was 7 × 10 −6 m while for the sorghum enzyme it was 3.7 × 10 −4 m. Inorganic phosphate proved to be an effective inhibitor of ADPglucose synthesis. The I 0.5 (conc of inhibitor that gave 50% inhibition of activity for the various leaf enzymes varied from 2 × 10 −5 m (barley) to 1.9 × 10 −4 m (sorghum). This inhibition was reversed or antagonized by the activator 3-phosphoglycerate. These results form the basis for an hypothesis of the regulation of leaf starch biosynthesis.

Starch phosphorylase (EC 2.4.1.1) and ADPglucose pyrophosphorylase (EC 2.7.7.27) activities can be measured very accurately and quickly using a recording pH meter. Activities less than 0.28 nmol/min are readily measured.

The ADPglucose pyrophosphorylases from Rhodopseudomonas sphaeroides and Rhodopseudomonas gelatinosa are activated by fructose-6-phosphate, pyruvate and fructose-1,6 biophosphate-P2. The effects of the activators are to increase significantly the Vmax of ADPglucose synthesis and to lower the S0.5 values (concentration of substrates giving 50% maximal velocity) for ATP and MgCl2. The R. sphaeroides enzyme is inhibited by Pi while the R. gelatinosa enzyme is inhibited by AMP as well as by Pi. The interaction between inhibitor and activator is complex. At very low concentrations of activator the enzyme is more sensitized to inhibition. However, at higher concentrations of activator there is a decrease in the sensitivity of the enzyme towards inhibition. The findings are discussed with respect to glycogen synthesis in these microorganisms and may be related to findings that indicate that Rhodopseudomonads have the ability to degrade sugars via the Entner-Duodoroff or Embden-Meyerhoff pathways.

Soluble ADPglucose-alpha-glucan 4-alpha-glucosyltransferase (starch synthetase), ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and phosphorylase were assayed in extracts from developing kernels of maize (Zea mays). Normal, waxy and amylose-extender maize at stages of development ranging from 8 days to 28 days after pollination were studied. Shrunken-4 maize at the 22-day stage was also studied. There is adequate activity of both ADPglucose pyrophosphorylase and starch synthetase at all stages of development to account for the synthesis of starch. Thus all starch could be synthesized via the ADPglucose pathway. High levels of UDPglucose pyrophosphorylase and of phosphorylase activities were also found at all stages of development. The possible role of phosphorylase in starch synthesis could not be discounted. The levels of phosphorylase, ADPglucose pyrophosphorylase, starch synthetase, and UDPglucose pyrophosphorylase activities in shrunken-4 kernels were about 20 to 40% of that found in normal maize kernels. It appears that the mutation in shrunken-4 affects the activities of more than one enzyme. The defective starch synthesis seen in this mutant could be due to the low activities of ADPglucose pyrophosphorylase and starch synthetase rather than the low activity of phosphorylase.