Masayama Atsushi, Takahashi Tetsuya, Tsukada Kaori, Nishikawa Seigo, Takahashi Rie, Adachi Masaatsu, Koga Kazushi, Suzuki Atsuo, Yamane Takashi, Nakano Hideo, Iwasaki Yugo
Laboratory of Molecular Biotechnology, Department of Bioengineering Sciences, Graduate School of Bioagricultural Science, Nagoya University, Nagoya 464-8601, Japan.
Chembiochem. 2008 Apr 14;9(6):974-81. doi: 10.1002/cbic.200700528.
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.
通过定点饱和诱变改变了抗生链霉菌磷脂酶D（PLD）的底物特异性，使其能够合成磷脂酰肌醇（PI）。在pld基因中对应于可能参与底物识别的三个氨基酸残基的位置引入突变，并在大肠杆菌BL21（DE3）中表达突变基因。对约10,000个菌落进行PI合成活性的高通量筛选，鉴定出25种PI合成突变型PLD。选择其中一种突变酶进行进一步分析。用高效液相色谱-质谱联用仪（HPLC-MS）和核磁共振（NMR）分析了用突变酶合成的PI的结构。发现突变酶产生了PI结构异构体的混合物，其磷脂酰基连接在肌醇环的不同位置。突变型PLD的磷脂酰胆碱水解活性远低于野生型酶。该突变酶能够使各种环己醇进行转磷脂酰基反应，更倾向于体积较大的化合物。这是链霉菌PLD底物特异性改变及PI合成的首个实例。
