Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis.
The substrate specificity of a phospholipase D (PLD) from Streptomyces antibioticus was altered by site-directed saturation mutagenesis, so that it was able to synthesize phosphatidylinositol (PI). Mutations were introduced in the pld gene at the positions corresponding to three amino acid residues that might be involved in substrate recognition, and the mutated genes were expressed in Escherichia coli BL21 (DE3). High-throughput screening of approximately 10,000 colonies for PI-synthesizing activity identified 25 PI-synthesizing mutant PLDs. One of these mutant enzymes was chosen for further analysis. The structure of the PI synthesized with the mutant enzyme was analyzed by HPLC-MS and NMR. It was found that the mutant enzyme generated a mixture of structural isomers of PIs with the phosphatidyl groups connected at different positions of the inositol ring. The phosphatidylcholine-hydrolyzing activity of the mutant PLD was much lower than that of the wild-type enzyme. The mutant enzyme was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds. This is the first example of alteration of the substrate specificity of PLD and of PI synthesis by Streptomyces PLD.
Masayama
Atsushi
A
Laboratory of Molecular Biotechnology, Department of Bioengineering Sciences, Graduate School of Bioagricultural Science, Nagoya University, Nagoya 464-8601, Japan.
Takahashi
Tetsuya
T
Tsukada
Kaori
K
Nishikawa
Seigo
S
Takahashi
Rie
R
Adachi
Masaatsu
M
Koga
Kazushi
K
Suzuki
Atsuo
A
Yamane
Takashi
T
Nakano
Hideo
H
Iwasaki
Yugo
Y
D001665:Binding Sites; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010713:Phosphatidylcholines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000096:biosynthesis*; D010739:Phospholipase D / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D013303:Streptomyces antibioticus / Q000201:enzymology*; D013379:Substrate Specificity / Q000235:genetics
D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D010739:Phospholipase D; C017251:1,2-oleoylphosphatidylcholine