In the present study, we cloned a gene, designated bpsA, which encodes a single module type non-ribosomal peptide synthetase (NRPS) from a D-cycloserine (DCS)-producing Streptomyces lavendulae ATCC11924. A putative oxidation domain is significantly integrated into the adenylation domain of the NRPS, and the condensation domain is absent from the module. When S. lividans was transformed with a plasmid carrying bpsA, the transformed cells produced a blue pigment, suggesting that bpsA is responsible for the blue pigment synthesis. However, to produce the blue pigment in Escherichia coli, the existence of the 4-phosphopantetheinyl transferase (PPTase) gene from Streptomyces was necessary, in addition to bpsA. The chemical structure of the pigment was determined as 5,5-diamino-4,4-dihydroxy-3,3-diazadiphenoquinone-(2,2), called indigoidine. The bpsA gene product, designated BPSA, was overproduced in an E. coli host-vector system and purified to homogeneity, demonstrating that the recombinant enzyme prefers L-Gln as a substrate. The in vitro experiment using L-Gln also showed that the blue pigment was formed by the purified BPSA only when the enzyme was phosphopantetheinylated by adding a Streptomyces PPTase purified from E. coli cells. Each site-directed mutagenesis experiment of Lys 598 , Tyr 601 , Ser 603 , and Tyr 608 , which are seen in the oxidation domain of BPSA, suggests that these residues are essential for the binding of FMN to the protein and the synthesis of the blue pigment.
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