title: Directing positional specificity in enzymatic synthesis of bioactive 1-phosphatidylinositol by protein engineering of a phospholipase D.
abstract: Phosphatidylinositol (PI) holds a potential of becoming an important dietary supplement due to its effects on lipid metabolism in animals and humans manifested as a decrease of the blood cholesterol and lipids, and relief of the metabolic syndrome. To establish an efficient, enzymatic system for PI production from phosphatidylcholine and myo-inositol as an alcohol acceptor, our previous study started with the wild-type Streptomyces antibioticus phospholipase D (SaPLD) as a template for generation of PI-synthesizing variants by saturation mutagenesis targeting positions involved in acceptor accommodation, W187, Y191, and Y385. The isolated variants generated PI as a mixture of positional isomers, among which only 1-PI exists in nature. Thus, the current study has focused to improve positional specificity of W187N/Y191Y/Y385R SaPLD (NYR) which generates PI as a mixture of 1-PI and 3-PI in the ratio of 76/24, by subjecting four residues of its acceptor-binding site to saturation mutagenesis. Subsequent screening pointed at NYR-186T and NYR-186L as the most improved variants producing PI with a ratio of 1-/3-PI = 93/7 and 87/13, respectively, at 37°C. Lowering the reaction temperature further improved the specificity of both variants to 1-/3-PI > 97/3 at 20°C with no change in total PI yield. Structure model analyses imply that G186T and G186L mutations increased rigidity of the acceptor-binding site, thus limiting the possible orientations of myo-inositol. The two newly isolated PLDs are promising for future application in large-scale 1-PI production.
authors.affiliation: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
authors.forename: Jasmina
authors.initials: J
authors.lastname: Damnjanović
authors.affiliation: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
authors.forename: Chisato
authors.initials: C
authors.lastname: Kuroiwa
authors.affiliation: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Tanaka
authors.affiliation: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Ishida
authors.affiliation: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Nakano
authors.affiliation: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan. iwasaki@agr.nagoya-u.ac.jp.
authors.forename: Yugo
authors.initials: Y
authors.lastname: Iwasaki
mesh_terms: D019943:Amino Acid Substitution; D007294:Inositol / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010739:Phospholipase D / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013303:Streptomyces antibioticus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D007294:Inositol; D010739:Phospholipase D
keywords: 1-phosphatidylinositol; phospholipase D; positional specificity