Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana

PCR amplification of cDNA prepared from poly(A)⁺ RNA from aerial parts of Arabidopsis thaliana, using degenerate nucleotide primers based on conserved regions between the large and small subunits of ADP-glucose pyrophosphorylase (AGP), yielded four different cDNAs of ca. 550 nucleotides each. Based on derived amino acid sequences, the identities between the clones varied from 49 to 69%. Sequence comparison to previously published cDNAs for AGP from various species and tissues has revealed that three of the amplified cDNAs (ApL1, ApL2 and ApL3) correspond to the large subunit of AGP, and one cDNA (ApS) encodes the small subunit of AGP. Both ApL1 and ApS were subsequently found to be present in a cDNA library made from Arabidopsis leaves. All four PCR products are encoded by single genes, as found by genomic Southern analysis.     

Crystal structure of potato tuber ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase catalyzes the first committed and rate-limiting step in starch biosynthesis in plants and glycogen biosynthesis in bacteria. It is the enzymatic site for regulation of storage polysaccharide accumulation in plants and bacteria, being allosterically activated or inhibited by metabolites of energy flux. We report the first atomic resolution structure of ADP-glucose pyrophosphorylase. Crystals of potato tuber ADP-glucose pyrophosphorylase alpha subunit were grown in high concentrations of sulfate, resulting in the sulfate-bound, allosterically inhibited form of the enzyme. The N-terminal catalytic domain resembles a dinucleotide-binding Rossmann fold and the C-terminal domain adopts a left-handed parallel beta helix that is involved in cooperative allosteric regulation and a unique oligomerization. We also report structures of the enzyme in complex with ATP and ADP-glucose. Communication between the regulator-binding sites and the active site is both subtle and complex and involves several distinct regions of the enzyme including the N-terminus, the glucose-1-phosphate-binding site, and the ATP-binding site. These structures provide insights into the mechanism for catalysis and allosteric regulation of the enzyme.     

Comparison of the primary sequences of two potato tuber ADP-glucose pyrophosphorylase subunits

Near-full-length cDNA clones to the small and large subunit of the heterotetrameric potato tuber ADP-glucose pyrophosphorylase have been isolated and characterized. The missing amino terminal sequence of the small subunit has also been elucidated from its corresponding genomic clone. Primary sequence comparisons revealed that each potato subunit had less identity to each other than to their homologous subunit from other plants. It also appeared that the smaller subunit is more conserved among the different plants and the larger subunit more divergent. Amino acid comparisons of both potato tuber sequences to theEscherichia coli ADP-glucose pyrophosphorylase sequence revealed conserved regions important for both catalytic and allosteric function of the bacterial enzyme.     

Isolation and nucleotide sequences of cDNA clones encoding ADP-glucose pyrophosphorylase polypeptides from wheat leaf and endosperm

A cDNA clone (WL : AGA.1) encoding wheat leaf ADP-glucose pyrophosphorylase has been isolated from a gt11 expression library, by immunological screening with anti-spinach leaf ADP-glucose pyrophosphorylase serum. The WL : AGA.1 cDNA is 948 bp long and contains approximately 55% of the complete wheat leaf ADP-glucose pyrophosphorylase mRNA sequence, estimated from Northern blot experiments. A wheat endosperm cDNA library was subsequently constructed in gt11 and six clones hybridising to the cDNA insert of clone WL : AGA.1 were isolated. The longest of these wheat endosperm ADP-glucose pyrophosphorylase cDNAs, clone WE : AGA.7, is nearly full-length (1798 bp), indicated by Northern blot analysis of wheat endosperm mRNA and nucleotide sequence analysis.Southern hybridisation analysis and restriction enzyme mapping indicated that the wheat leaf and wheat endosperm ADP-glucose pyrophosphorylase cDNAs and genes are members of two distinct gene families. In addition, restriction enzyme mapping revealed polymorphism in the wheat endosperm ADP-glucose pyrophosphorylase cDNAs, indicating the existence of at least two wheat endosperm ADP-glucose pyrophosphorylase gene sub-families.Subsequent nucleotide sequence analysis indicates that there is approximately 55% identity between wheat leaf and wheat endosperm ADP-glucose pyrophosphorylase cDNAs. In contrast, members of each sub-family of endosperm cDNA, represented by clones WE : AGA.3 and WE : AGA.7, are 96% identical.     

Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) is one of the major enzymes involved in starch biosynthesis in higher plants. We report here the molecular cloning of two cDNAs encoding so far uncharacterized isoforms (AGP S2 and AGP S3) of the potato enzyme. Sequence analysis shows that the two polypeptides are more homologous to previously identified large subunit polypeptides from potato and other plant species than to small subunit isoforms. This observation suggests that AGP S2 and AGP S3 represent novel large subunit polypeptides. agpS2 is expressed in several tissues of the potato plant, including leaves and tubers. Expression was stronger in sink leaves than in source leaves, indicating developmental regulation. In leaves, agpS2 expression was induced 2- to 3-fold by exogenous sucrose; therefore, agpS2 represents a new sucrose-responsive gene of starch metabolism. Expression of agpS3 was restricted to tubers: no agpS3 expression could be seen in leaves of different developmental stages, or when leaves were incubated in sucrose. Therefore, agpS3 represents the only AGPase gene so far characterized from potato, which is not expressed in leaves. Conversely, all four AGPase isoforms known from potato are expressed in tubers.     

Contribution of sucrose synthase, ADP-glucose pyrophosphorylase and starch synthase to starch synthesis in developing pea seeds

Using genetic variability existing amongst nine pea genotypes (Pisum sativum L.), the biochemical basis of sink strength in developing pea seeds was investigated. Sink strength was considered to be reflected by the rate of starch synthesis (RSS) in the embryo, and sink activity in the seed was reflected by the relative rate of starch synthesis (RRSS). These rates were compared to the activities of three enzymes of the starch biosynthetic pathway [sucrose synthase (Sus), ADP-glucose pyrophosphorylase and starch synthase] at three developmental stages during seed filling (25, 50 and 75% of the dry seed weight). Complete sets of data collected during seed filling for the nine genotypes showed that, for all enzyme activities (expressed on a protein basis), only Sus in the embryo and seed coat was linearly and significantly correlated to RRSS. The contribution of the three enzyme activities to the variability in RSS and RRSS was evaluated by multiple regression analysis for the first two developmental stages. Only Sus activity in the embryo could explain, at least in part, the significant variability observed for both the RSS and the RRSS at each developmental stage. We conclude that Sus activity is a reliable marker of sink activity in developing pea seeds.     

Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm

Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken endosperms contained starch granules, whereas those in sugary endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice endosperm.     

Comparison of proteins of ADP-glucose pyrophosphorylase from diverse sources

Summary The primary structures of 11 proteins of ADP-glucose pyrophosphorylase are aligned and compared for relationships among them. These comparisons indicate that many domains are retained in the proteins from both the enteric bacteria and the proteins from angiosperm plants. The proteins from angiosperm plants show two main groups, with one of the main groups demonstrating two subgroups. The two main groups of angiosperm plant proteins are based upon the two subunits of the enzyme, whereas the subgroups of the large subunit group are based upon the tissue in which the particular gene had been expressed. Additionally, the small subunit group shows a slight but distinct division into a grouping based upon whether the protein is from a monocot or dicot source. Previous structure-function studies with the Escherichia coli enzyme have identified regions of the primary structure associated with the substrate binding site, the allosteric activator binding site, and the allosteric inhibitor binding site. There is conservation of the primary structure of the polypeptides for the substrate binding site and the allosteric activator binding site. The nucleotide sequences of the coding regions of the genes of 11 of these proteins are compared for relationships among them. This analysis indicates that the protein for the small subunit has been subject to greater selective pressure to retain a particular primary structure. Also, the coding region of the precursor gene for the small subunit diverged from the coding region of the precursor gene for the large subunits slightly prior to the divergence of the two coding regions of the genes for the two tissue-specific large subunit genes.Offprint requests to: J. Preiss     

Expression of a wheat ADP-glucose pyrophosphorylase gene during development of normal and water-stress-affected anthers

In wheat (Triticum aestivum L.), water deficit during meiosis in the microspore mother cells (MMCs) induces pollen abortion, resulting in the failure of fertilization and a reduction in grain set. In stressed plants, meiosis in MMCs proceeds normally but subsequent pollen development is arrested. Unlike normal pollen grains, which accumulate starch during the late maturation phase, stress-affected anthers contain pollen grains with little or no starch. Stress also alters the normal distribution of starch in the anther wall and connective tissue. To determine how starch biosynthesis is regulated within the developing anthers of stressed plants, we studied the expression of ADP-glucose pyrophosphorylase (AGP), which catalyzes the rate limiting step of starch biosynthesis. Two partial-length cDNAs corresponding to the large subunit of AGP were amplified by RT-PCR from anther RNA, and used as probes to monitor AGP expression in developing anthers of normal and water-stressed plants. These clones, WAL1 and WAL2, had identical deduced amino acid sequences and shared 96% sequence identity at the nucleic acid level. In normal anthers, AGP expression was biphasic, indicating that AGP expression is required for starch biosynthesis both during meiosis and later during pollen maturation. AGP expression in stressed anthers was not affected during the first phase of starch accumulation, but was strongly inhibited during the second phase. We conclude from these results that the reduced starch deposition later in the development of stressed pollen could be the result of a lower expression of AGP. However, this inhibition of AGP expression is unlikely to be the primary cause of male sterility because anatomical symptoms of pollen abortion are observed prior to the time when AGP expression is inhibited.     

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacteriumAnabaena sp. strain PCC 7120

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacteriumAnabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of theAnabaena ADPGlc PPase gene and its expression inEscherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48347 Da which is in agreement with the molecular mass determined by SDS-PAGE for theAnabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and theE. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in theAnabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in anE. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the nativeAnabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be theAnabaena enzyme.     

ADP-glucose pyrophosphorylase from potato tuber: site-directed mutagenesis of homologous aspartic acid residues in the small and large subunits

Asp142 in the homotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase) enzyme from Escherichia coli was demonstrated to be involved in catalysis of this enzyme [Frueauf, J.B., Ballicora, M.A. and Preiss J. (2001) J. Biol. Chem., 276, 46319-46325]. The residue is highly conserved throughout the family of ADP-Glc PPases, as well as throughout the super-family of sugar-nucleotide pyrophosphorylases. In the heterotetrameric ADP-Glc PPase from potato (Solanum tuberosum L.) tuber, the homologous residue is present in both the small (Asp145) and the large (Asp160) subunits. It has been proposed that the small subunit of plant ADP-Glc PPases is catalytic, while the large subunit is modulatory; however, no catalytic residues have been identified. To investigate the function of these conserved Asp residues in the ADP-Glc PPase from potato tuber, we used site-directed mutagenesis to introduce either an Asn or a Glu. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of ADP-Glc showed a significant decrease (more than four orders of magnitude) in the specific activity of the SD145NLwt, SD145NLD160N, and SD145NLD160E mutants, while the effect was smaller (approximately two orders of magnitude) with the SD145ELwt, SD145ELD160N, and SD145ELD160E mutants. By contrast, mutation of the large subunit alone did not affect the specific activity but did alter the apparent affinity for the activator 3-phosphoglycerate, showing two types of apparent roles for this residue in the different subunits. These results show that mutation of Asp160 of the large subunit does not affect catalysis, thus the large subunit is not catalytic, and that the negative charge of Asp145 in the small subunit is necessary for enzyme catalysis.     

Understanding the allosteric trigger for the fructose-1,6-bisphosphate regulation of the ADP-glucose pyrophosphorylase from Escherichia coli

ADP-glucose pyrophosphorylase is the enzyme responsible for the regulation of glycogen synthesis in bacteria. The enzyme N-terminal domain has a Rossmann-like fold with three neighbor loops facing the substrate ATP. In the Escherichia coli enzyme, one of those loops also faces the regulatory site containing Lys³⁹, a residue involved in binding of the allosteric activator fructose-1,6-bisphosphate and its analog pyridoxal-phosphate. The other two loops contain Trp¹¹³ and Gln⁷⁴, respectively, which are highly conserved among all the ADP-glucose pyrophosphorylases. Molecular modeling of the E. coli enzyme showed that binding of ATP correlates with conformational changes of the latter two loops, going from an open to a closed (substrate-bound) form. Alanine mutants of Trp¹¹³ or Gln⁷⁴ did not change apparent affinities for the substrates, but they became insensitive to activation by fructose-1,6-bisphosphate. By capillary electrophoresis we found that the mutant enzymes still bind fructose-1,6-bisphosphate, with similar affinity as the wild type enzyme. Since the mutations did not alter binding of the activator, they must have disrupted the communication between the regulatory and the substrate sites. This agrees with a regulatory mechanism where the interaction with the allosteric activator triggers conformational changes at the level of loops containing residues Trp¹¹³ and Gln⁷⁴.     

An assay for adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase

Adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5'-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S(0.5) determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.