Directed Mutagenesis, Ser-56 to Pro, ofBacillus subtilisPhosphatidylserine Synthase Drastically Lowers Enzymatic Activity and Relieves Amplification Toxicity inEscherichia coli

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Abstract

Amino acid residue 56 of the phosphatidylserine synthase of Bacillus subtilis was changed from Ser to Pro by using modified primers in PCR amplification of its structural gene, pssBS. When an Escherichia coli mutant lacking its own phosphatidylserine synthase harbored a plasmid carrying this allele, the Mn(2+)-requiring Bacillus-type synthase activity, as assayed in vitro, was at least six-fold lower than that with the wild-type pssBS gene and the cellular phosphatidylethanolamine content was similarly lowered, indicating that the altered region of the enzyme is critically important for its activity. In contrast to the E. coli counterpart, amplification of the wild-type Bacillus enzyme increased both the relative and absolute contents of phosphatidylethanolamine and impaired cell growth. However, amplification of the mutant enzyme of the same level was much less toxic, implying that E. coli cells are more sensitive to the unbalanced accumulation of phosphatidylethanolamine than that of the hydrophobic enzyme molecules. Possible roles of the conserved region of the enzyme in its activity and the wild-type phospholipid composition in the proper membrane function are discussed.

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