Engineered plant phosphorylase showing extraordinarily high affinity for various alpha-glucan molecules.
alpha-Glucan phosphorylases are characterized by considerable difference in substrate specificities, even though the primary structures are well conserved among the enzymes from microorganisms, plants, and animals. The higher plant phosphorylase isozyme designated as type L exhibits low affinity for a large, highly branched glucan (glycogen), presumably due to steric hindrance caused by a unique 78-residue insertion located beside the mouth of the active-site cleft, whereas another isozyme without the insertion (designated as type H) shows very high affinity for both linear and branched glucans. Using the recombinant type L isozyme from potato tuber as a starting framework and aiming at altering its substrate specificity, we have genetically engineered the 78-residue insertion and its flanking regions. Firstly, removal of the insertion and connection of the newly formed C- and N-terminals yielded a totally inactive enzyme, although the protein was produced in Escherichia coli cells in a soluble form. Secondly, a chimeric phosphorylase, in which the 78-residue insertion and its flanking regions are replaced 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, 5574-5581), but when two and four unconserved residues in the N-terminal flanking region of the chimeric phosphorylase were mutated back to those of the type L isozyme, the resulting mutants showed significantly lowered affinity for substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
Mori
H
H
Institute of Scientific and Industrial Research, Osaka University, Japan.
Tanizawa
K
K
Fukui
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D006003:Glycogen / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D016296:Mutagenesis; D006005:Phosphorylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006003:Glycogen; D006005:Phosphorylases