-Poly-L-lysine (-PL) is produced by Streptomyces albulus NBRC14147 as a secondary metabolite and can be detected only when the fermentation broth has an acidic pH during the stationary growth phase. Since strain NBRC14147 produces -PL-degrading enzymes, the original chain length of the -PL polymer product synthesized by -PL synthetase (Pls) is unclear. Here, we report on the identification of the gene encoding the -PL-degrading enzyme (PldII), which plays a central role in -PL degradation in this strain. A knockout mutant of the pldII gene was found to produce an -PL of unchanged polymer chain length, demonstrating that the length is not determined by -PL-degrading enzymes but rather by Pls itself and that the 25 to 35 L-lysine residues of -PL represent the original chain length of the polymer product synthesized by Pls in vivo. Transcriptional analysis of pls and a kinetic study of Pls further suggested that the Pls catalytic function is regulated by intracellular ATP, high levels of which are required for full enzymatic activity. Furthermore, it was found that acidic pH conditions during -PL fermentation, rather than the inhibition of the -PL-degrading enzyme, are necessary for the accumulation of intracellular ATP.
b-Poly-L-lysine (-PL), consisting of 25 to 35 L-lysine residues with linkages between the ␣-carboxyl groups and -amino groups, is produced by Streptomyces albulus NBRC14147. -PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates -PL chain length diversity (25-to 35-mer), but the processes that control the chain length of -PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the -PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of -PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of -PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of -PL.
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