Cloning and characterization of a Streptomyces single module type non-ribosomal peptide synthetase catalyzing a blue pigment synthesis.
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.
Takahashi
Hitoshi
H
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical Sciences, Hiroshima University, Japan.
Kumagai
Takanori
T
Kitani
Kyoko
K
Mori
Miwako
M
Matoba
Yasuyuki
Y
Sugiyama
Masanori
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010453:Peptide Synthases / Q000235:genetics*; D010858:Pigmentation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013302:Streptomyces / Q000235:genetics*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
D001426:Bacterial Proteins; C082662:phosphopantetheinyl transferase; D014443:Tyrosine; D012694:Serine; D017855:Transferases (Other Substituted Phosphate Groups); D010453:Peptide Synthases; D008239:Lysine; D010100:Oxygen