The Gene Structure of Starch Phosphorylase from Sweet Potato

a-Glucan phosphorylases are of key importance among the enzymes controlling the entry of storage polysaccharides into the glycolytic pathway in microbes, animals, and plants. The enzymes catalyze the reversible phosphorolysis of cu-l,4-glucosidic linkages in glucan substrates. Among SP from higher plants, those from potato are best studied. They are classified into L and H types depending on their low and high affinities toward branched glucans, respectively (Nakano et al., 1989). The L type is larger than the H type in having a 78-amino acid residue insertion at the central position, causing the molecule to show a low homology toward phosphorylases from other sources (Nakano and Fukui, 1986; Nakano et al., 1989; Lin et al., 1991; Mori et al., 1991). The presence of the extra peptide was suggested to affect the substrate affinity (Nakano and Fukui, 1986), and this was proven to be true by a protein engineering approach (Mori et al., 1993). Animal glycogen phosphorylases are endowed with both covalent and allosteric regulatory mechanisms (Fukui et al., 1982; Hwang and Fletterick, 1986) and their structurefunction relationship has been established, but neither such functions nor the corresponding structures are known for either Lor H-type plant phosphorylases. Previously, we postulated that SP in the growing sweet potato (Ipomoea batatus L.) root could be involved in a starch synthesis pathway. Gel electrophoresis analysis of sweet potato SP always gave multiple bands, which indicated the presence of subunits with different proteolytic susceptibility at the midchain 78-residue segment. Determination of amino acid sequence was hindered by blocking of the N terminus and the difficulty of obtaining intact pure peptide. We have cloned and sequenced a cDNA encoding this L-type isozyme (Lin et al., 1991). The cDNA encodes a 955-residue polypeptide that has 81 % homology toward the L-type potato enzyme (916 residues plus a 50-residue putative transit peptide). Higher divergence of the two enzymes was found in about 70 residues of the N termini, including a putative transit peptide, and in the midchain 78-residue insert. Very high similarity between the potato