Function of the maize (Zea mays) gene sugary1 (su1) is required for normal starch biosynthesis in endosperm. Homozygous su1-mutant endosperms accumulate a highly branched polysaccharide, phytoglycogen, at the expense of the normal branched component of starch, amylopectin. These data suggest that both branched polysaccharides share a common precursor, and that the product of the su1 gene, designated SU1, participates in kernel starch biosynthesis. SU1 is similar in sequence to ␣-(136) glucan hydrolases (starch-debranching enzymes [DBEs]). Specific antibodies were produced and used to demonstrate that SU1 is a 79-kD protein that accumulates in endosperm coincident with the time of starch biosynthesis. Nearly full-length SU1 was expressed in Escherichia coli and purified to apparent homogeneity. Two biochemical assays confirmed that SU1 hydrolyzes ␣-(136) linkages in branched polysaccharides. Determination of the specific activity of SU1 toward various substrates enabled its classification as an isoamylase. Previous studies had shown, however, that su1-mutant endosperms are deficient in a different type of DBE, a pullulanase (or R enzyme). Immunoblot analyses revealed that both SU1 and a protein detected by antibodies specific for the rice (Oryza sativa) R enzyme are missing from su1-mutant kernels. These data support the hypothesis that DBEs are directly involved in starch biosynthesis.Starch is a reserve carbohydrate that accumulates in the storage organs of many higher plants. This storage compound consists of a mixture of two Glc homopolymers, amylopectin and amylose, in which linear chains are formed via ␣-(13 4) glucosyl linkages and branches are introduced by ␣-(13 6) glucosyl linkages. Starch synthesis in maize (Zea mays) occurs within the amyloplasts of endosperm cells during kernel development via the concerted actions of ADP-Glc pyrophosphorylase, starch synthases, and starch-branching enzymes (for reviews, see Preiss, 1991;Hannah et al., 1993;Martin and Smith, 1995;Nelson and Pan, 1995; Preiss and Sivak, 1996; Smith et al., 1996). In addition, selective removal of branch linkages by DBEs is proposed to play an essential role in the final determination of amylopectin structure (Ball et al., 1996).Physical and chemical analyses of granular starch have led to a widely accepted model for amylopectin structure called the "cluster model," in which amorphous and crystalline regions alternate with a defined periodicity (for reviews, see French, 1984;Manners, 1989;Jenkins et al., 1993). Within amylopectin the crystalline component is composed of parallel arrays of linear chains packed tightly in double helices. Branch linkages, which account for approximately 5% of the glucosyl linkages in amylopectin, are located at the root of each cluster in the amorphous region. This periodic clustering of branches allows for the alignment of the intervening linear chains and their dense packing into crystalline regions, thus providing an efficient mechanism for nutrient storage. Undoubtedly, the enzymatic processes requir...

This study identified and purified specific isoamylase-and pullulanase-type starch-debranching enzymes (DBEs) present in developing maize (Zea mays L.) endosperm. The cDNA clone Zpu1 was isolated based on its homology with a rice (Oryza sativa L.) cDNA coding for a pullulanase-type DBE. Comparison of the protein product, ZPU1, with 18 other DBEs identified motifs common to both isoamylase-and pullulanase-type enzymes, as well as classspecific sequence blocks. Hybridization of Zpu1 to genomic DNA defined a single-copy gene, zpu1, located on chromosome 2. Zpu1 mRNA was abundant in endosperm throughout starch biosynthesis, but was not detected in the leaf or the root. Anti-ZPU1 antiserum specifically recognized the approximately 100-kD ZPU1 protein in developing endosperm, but not in leaves. Pullulanase-and isoamylase-type DBEs were purified from extracts of developing maize kernels. The pullulanase-type activity was identified as ZPU1 and the isoamylase-type activity as SU1. Mutations of the sugary1 (su1) gene are known to cause deficiencies of SU1 isoamylase and a pullulanase-type DBE. ZPU1 activity, protein level, and electrophoretic mobility were altered in su1-mutant kernels, indicating that it is the affected pullulanase-type DBE. The Zpu1 transcript levels were equivalent in nonmutant and su1-mutant kernels, suggesting that coordinated regulation of ZPU1 and SU1 occurs posttranscriptionally.Amylopectin is a branched Glc polymer that is a major constituent of plant starch granules and is the primary determinant of their structural and physical properties. The spatial positioning of ␣(136) glycosidic bonds, i.e. branch linkages, is a critical aspect of the three-dimensional structure of amylopectin (Gallant et al., 1997). Branch linkages are introduced by the actions of starch branching enzymes and are hydrolyzed by the actions of DBEs (for recent reviews, see Preiss and Sivak, 1996;Smith et al., 1997). Mutations that result in DBE deficiencies, such as the sugary1 (su1) mutations of maize (Zea mays L.) and rice (Pan and Nelson, 1984; James et al., 1995;Nakamura et al., 1996b;Rahman et al., 1998), alter the number and spatial distribution of branches in amylopectin. Therefore, DBEs are believed to be involved in branch-pattern determination, possibly providing an editing function (Ball et al., 1996).Two classes of DBEs have been identified in plants that are distinguishable by their substrate specificities (Lee and Whelan, 1971; Doehlert and Knutson, 1991). "Isoamylasetype" DBEs cleave ␣(136) branch linkages in amylopectin and glycogen, but do not hydrolyze the chemically identical bonds in pullulan, an ␣(136)-linked maltotriose polymer. In contrast, "pullulanase-type" DBEs, also referred to as R-enzymes or limit-dextrinases (Manners, 1997), readily hydrolyze ␣(136) linkages of pullulan or amylopectin, but have little activity toward glycogen. Biochemical fractionation experiments identified both isoamylase-and pullulanase-type DBE activities in developing maize kernels during the starch biosynthetic period (...

APS reductase from Pseudomonas aeruginosa has been shown to contain a [4Fe-4S] cluster. Thiol determinations and site-directed mutagenesis studies indicate that the single [4Fe-4S] cluster contains only three cysteine ligands, instead of the more typical arrangement in which clusters are bound to the protein by four cysteines. Resonance Raman studies in the Fe-S stretching region are also consistent with the presence of a redox-inert [4Fe-4S](2+) cluster with three cysteinate ligands and indicate that the fourth ligand is likely to be an oxygen-containing species. This conclusion is supported by resonance Raman and electron paramagnetic resonance (EPR) evidence for near stoichiometric conversion of the cluster to a [3Fe-4S](+) form by treatment with a 3-fold excess of ferricyanide. Site-directed mutagenesis experiments have identified Cys139, Cys228, and Cys231 as ligands to the cluster. The remaining two cysteines present in the enzyme, Cys140 and Cys256, form a redox-active disulfide/dithiol couple (E(m) = -300 mV at pH 7.0) that appears to play a role in the catalytic mechanism of the enzyme.

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APS reductase from Pseudomonas aeruginosa has been shown to form a disulfide-linked adduct with mono-cysteine variants of Escherichia coli thioredoxin and Chlamydomonas reinhardtii thioredoxin h1. These adducts presumably represent trapped versions of the intermediates formed during the catalytic cycle of this thioredoxin-dependent enzyme. The oxidation-reduction midpoint potential of the disulfide bond in the P. aeruginosa APS reductase/C. reinhardtii thioredoxin h1 adduct is -280 mV. Site-directed mutagenesis and mass spectrometry have identified Cys256 as the P. aeruginosa APS reductase residue that forms a disulfide bond with Cys36 of C. reinhardtii TRX h1 and Cys32 of E. coli thioredoxin in these adducts. Spectral perturbation measurements indicate that P. aeruginosa APS reductase can also form a non-covalent complex with E. coli thioredoxin and with C. reinhardtii thioredoxin h1. Perturbation of the resonance Raman and visible-region absorbance spectra of the APS reductase [4Fe-4S] center by either APS or the competitive inhibitor 5'-AMP indicates that both the substrate and product bind in close proximity to the cluster. These results have been interpreted in terms of a scheme in which one of the redox-active cysteine residues serves as the initial reductant for APS bound at or in close proximity to the [4Fe-4S] cluster.

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