Mutational analysis of the three tandem domains of ε-poly-L-lysine synthetase catalyzing the L-lysine polymerization reaction.

ε-Poly-l-lysine (ε-PL) synthetase (Pls) is a nonribosomal peptide synthetase (NRPS)-like enzyme with three tandem domains to catalyze the l-lysine polymerization reaction. Mutational analysis of the three tandem domains demonstrated that the interconnected action of all three domains is essential for the enzyme activity.

A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis.

The streptothricin (ST) antibiotics, produced by Streptomyces bacteria, contain L-β-lysine ((3S)-3,6-diaminohexanoic acid) oligopeptides as pendant chains. Here we describe three unusual nonribosomal peptide synthetases (NRPSs) involved in ST biosynthesis: ORF 5 (a stand-alone adenylation (A) domain), ORF 18 (containing thiolation (T) and condensation (C) domains) and ORF 19 (a stand-alone A domain). We demonstrate that ST biosynthesis begins with adenylation of L-β-lysine by ORF 5, followed by transfer to the T domain of ORF 18.

Development of a recombinant ε-poly-L-lysine synthetase expression system to perform mutational analysis.

ε-Poly-L-lysine (ε-PL) synthetase (Pls), which is a membrane protein with adenylation and thiolation domains characteristic of the nonribosomal peptide synthetases, catalyzes polymerization of L-lysine molecules (25-mer to 35-mer). Here, we report on the development of a recombinant Pls expression system that allowed us to perform a site-directed mutational analysis.

Mechanism of epsilon-poly-L-lysine production and accumulation revealed by identification and analysis of an epsilon-poly-L-lysine-degrading enzyme.

Epsilon-poly-L-lysine (epsilon-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 epsilon-PL-degrading enzymes, the original chain length of the epsilon-PL polymer product synthesized by epsilon-PL synthetase (Pls) is unclear. Here, we report on the identification of the gene encoding the epsilon-PL-degrading enzyme (PldII), which plays a central role in epsilon-PL degradation in this strain.

Acid phosphatase/phosphotransferases from enteric bacteria.

We have investigated the enzymatic phosphorylation of nucleosides and found that Morganella morganii phoC acid phosphatase exhibits regioselective pyrophosphate (PP(i))-nucleoside phosphotransferase activity. In this study, we isolated genes encoding an acid phosphatase with regioselective phosphotransferase activity (AP/PTase) from Providencia stuartii, Enterobacter aerogenes, Escherichia blattae and Klebsiella planticola, and compared the primary structures and enzymatic characteristics of these enzymes with those of AP/PTase (PhoC acid phosphatase) from M. morganii.