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Directing positional specificity in enzymatic synthesis of bioactive 1‐phosphatidylinositol by protein engineering of a phospholipase D
Author(s) -
Damnjanović Jasmina,
Kuroiwa Chisato,
Tanaka Hidetoshi,
Ishida Ken,
Nakano Hideo,
Iwasaki Yugo
Publication year - 2016
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25697
Subject(s) - phosphatidylinositol , enzyme , biochemistry , phospholipase c , chemistry , phospholipase , phospholipase d , protein engineering , phosphoinositide phospholipase c , signal transduction
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. Biotechnol. Bioeng. 2016;113: 62–71. © 2015 Wiley Periodicals, Inc.