Biomaterial Database

Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis. 2008

Atsushi Masayama, and Tetsuya Takahashi, and Kaori Tsukada, and Seigo Nishikawa, and Rie Takahashi, and Masaatsu Adachi, and Kazushi Koga, and Atsuo Suzuki, and Takashi Yamane, and Hideo Nakano, and Yugo Iwasaki

Laboratory of Molecular Biotechnology, Department of Bioengineering Sciences, Graduate School of Bioagricultural Science, Nagoya University, Nagoya 464-8601, Japan.

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.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008958	Models, Molecular	Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures.	Molecular Models,Model, Molecular,Molecular Model
D009154	Mutation	Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.	Mutations
D010713	Phosphatidylcholines	Derivatives of PHOSPHATIDIC ACIDS in which the phosphoric acid is bound in ester linkage to a CHOLINE moiety.	Choline Phosphoglycerides,Choline Glycerophospholipids,Phosphatidyl Choline,Phosphatidyl Cholines,Phosphatidylcholine,Choline, Phosphatidyl,Cholines, Phosphatidyl,Glycerophospholipids, Choline,Phosphoglycerides, Choline
D010716	Phosphatidylinositols	Derivatives of phosphatidic acids in which the phosphoric acid is bound in ester linkage to the hexahydroxy alcohol, myo-inositol. Complete hydrolysis yields 1 mole of glycerol, phosphoric acid, myo-inositol, and 2 moles of fatty acids.	Inositide Phospholipid,Inositol Phosphoglyceride,Inositol Phosphoglycerides,Inositol Phospholipid,Phosphoinositide,Phosphoinositides,PtdIns,Inositide Phospholipids,Inositol Phospholipids,Phosphatidyl Inositol,Phosphatidylinositol,Inositol, Phosphatidyl,Phosphoglyceride, Inositol,Phosphoglycerides, Inositol,Phospholipid, Inositide,Phospholipid, Inositol,Phospholipids, Inositide,Phospholipids, Inositol
D010739	Phospholipase D	An enzyme found mostly in plant tissue. It hydrolyzes glycerophosphatidates with the formation of a phosphatidic acid and a nitrogenous base such as choline. This enzyme also catalyzes transphosphatidylation reactions. EC 3.1.4.4.	Lecithinase D,Phosphatidylcholine Phosphohydrolase
D011487	Protein Conformation	The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).	Conformation, Protein,Conformations, Protein,Protein Conformations
D006868	Hydrolysis	The process of cleaving a chemical compound by the addition of a molecule of water.
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D013303	Streptomyces antibioticus	An actinomycete from which the antibiotic OLEANDOMYCIN is obtained.