ENGINEERED PLANT PHOSPHORYLASE SHOWING EXTRAORDINARILY HIGH-AFFINITY FOR VARIOUS ALPHA-GLUCAN MOLECULES

Alpha-Glucan phosphorylases are characterized by considerable differen ce in substrate specificities, even though the primary structures are well conserved among the enzymes from microorganisms, plants, and anim als. The higher plant phosphorylase isozyme designated as type L exhib its low affinity for a large, highly branched glucan (glycogen), presu mably due to steric hindrance caused by a unique 78-residue insertion located beside the mouth of the active-site cleft, whereas another iso zyme without the insertion (designated as type H) shows very high affi nity for both linear and branched glucans. Using the recombinant type L isozyme from potato tuber as a starting framework and aiming at alte ring its substrate specificity, we have genetically engineered the 78- residue insertion and its flanking regions. Firstly, removal of the in sertion and connection of the newly formed C- and N-terminals yielded a totally inactive enzyme, although the protein was produced in Escher ichia coli cells in a soluble form. Secondly, a chimeric phosphorylase , in which the 78-residue insertion and its flanking regions are repla ced by the corresponding region of the type H isozyme, has been shown to exhibit high affinity for branched glucans (Mori, H., Tanizawa, K., & Fukui, T., 1993, J. Biol. Chem. 268, 5574-558 1), but when two and four unconserved residues in the N-terminal flanking region of the chi meric phosphorylase were mutated back to those of the type L isozyme, the resulting mutants showed significantly lowered affinity for substr ates. Finally, a chimeric phosphorylase, in which a 112-residue sequen ce of the rabbit muscle enzyme involving the glycogen-storage site was substituted for the 78-residue insertion and its flanking regions of the type L isozyme, showed extraordinarily high affinity for a variety of substrates including not only large, branched glucans but also sma ll, linear ones.