Abstract
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.
Highlights
Many peptide antibiotics are known to be synthesized by non-ribosomal peptide synthetases (NRPSs)
NRPSs, which are commonly found in microorganisms, are very large proteins containing sets of modules, each of which consists of various functional domains such as adenylation (A), condensation (C), cyclization (Cy), thiolation (T), and thioesterase (TE) domains [3]
In this study, during our attempt to clone DCS biosynthesis genes from a DCS-producing S. lavendulae ATCC11924 by the suppression subtractive hybridization method, which is a costeffective and powerful technique for the isolation of speciesspecific DNA sequences from closely related microorganisms [7, 8], we unexpectedly found that a DCS producer-originated gene, designated bpsA, encodes a protein classified into the Streptomyces antibiotic regulatory protein; T-domain, thiolation domain; TE-domain, thioesterase domain; THF, tetrahydrofuran; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; BPSA, blue pigment synthetase A
Summary
Bacterial Strains, Plasmids, and Growth Conditions—S. lavendulae ATCC11924 and S. lavendulae JCM4055 are a DCS producer and a DCS-non-producer, respectively. The assay solution (100 l) contained 300 nM His6-tagged protein, 5 mM ATP, 1.72 M [32P]PPi (1 Ci, 60 Ci/mmol; PerkinElmer), 1 mM PPi, 1 mM MgCl2, 0.1 mM EDTA, 0.5 mM amino acid, and 20 mM Tris-HCl, pH 7.8. 15 min after addition of L-[14C]Gln, the reaction was quenched tein deduced from the nucleotide sequence had a 70% similarity with 0.8 ml of 10% cold trichloroacetic acid containing 2% to a putative regulatory protein of actinorhodin-producing bovine serum albumin. In Vitro Synthesis of the Blue Pigment—To phosphopanteth- secondary metabolites [11], a 6-kb BamHI-digested DNA einylate BPSA, a solution (1.4 ml) containing 660 nM BPSA, 810 fragment was newly cloned from the genome of S. The protein, encoded by lated BPSA was subjected to SDS-PAGE, and the correspond- orfB, consists of 1282 amino acids with a calculated molecular ing band was cut off from the gel. LTFREL [43–48] LNAGAAYLPLS [88–98] LAYVIYTSGSTGKPKG [166–181] FDAA [216–219] NVYGPTE [309–315] GELYIGGIQLARGYL [365–379] YKTGDL [404–409] GRADNQVKLRGYRVELDEIS [422–441] LPDYMIP [892–898] NGKIDV [912–917] DDFFESGGNSL [963–973] GYSFG (1100–1104)
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