Functional analysis of fengycin synthetase FenD

Nonribosomal Synthesis of Fengycin on an Enzyme Complex Formed by Fengycin Synthetases

“…Proteins that were bound to the beads were purified according to the method described above and dialyzed against a buffer that contained 50 mM Tris-HCl, pH 7.8, 0.5 mM Na 4 P 2 O 7 , and 20% sucrose. Reconstituted fengycin synthetase complexes with FenB-dTE (2.6 g) were added to a reaction mixture that contained 2 mM MgCl 2 ; 2 mM 1,4-dithiothreitol; 2 mM ATP; 2 mM EDTA; 20 mM Mes-Hepes, pH 7.5; 100 14 ]tyrosine (473 mCi/mmol), and L- [C 14 ]proline (252 mCi/mmol) (Amersham Biosciences). The reaction mixture (200 l) was incubated at 25°C for 30 min.…”

“…After the complex had been purified by Ni 2ϩ -NTA-agarose affinity chromatography, the enzyme complex was added to a reaction mixture that contained the seven substrate amino acids that were involved in fengycin synthesis, including 14 C-labeled L-Orn, L-Tyr, and L-Pro. After the reaction completed, urea was added to the reaction mixture to dissociate the enzymes from FenB-dTE.…”

“…Subsequently, a transpeptidation process transfers the activated amino acid in the initiating module, FenC1, in the initiating enzyme, FenC, to the activated amino acid at the thiolation domain in the next module, FenC2, ultimately forming a dipeptide, L-Glu-L-Orn, on FenC (5,20,21). In the next step, the dipeptide synthesized on FenC is translocated to FenD; during this process, L-Orn is racemized to D-Orn (22) and linked with L-Tyr activated by the FenD1 module (14,21,23). This process continues from one module to another and from one peptide synthetase to another peptide synthetase until the elongating peptide chain reaches FenB (12), which contains a thioesterase domain that terminates peptide synthesis and releases the peptide chain from the enzyme (24,25).…”

“…These enzymes typically contain from one to several amino acid activation modules that are about 1000 amino acids long, activating a specific amino acid for peptide synthesis (6 -10). In each module, an adenylation domain of ϳ550 amino acids recognizes and adenylates a specific amino acid (5,(11)(12)(13)(14). Following adenylation, the amino acid forms a thioester bond with the cofactor 4Ј-phosphopantetheine bound to the thiolation domain located C-terminal to the adenylation domain in the module (15)(16)(17)(18)(19).…”

Activation of the Promoter of the Fengycin Synthetase Operon by the UP Element

“…These enzymes interlock to form a chain in the order FenC-FenD-FenE-FenAFenB in interactions between the C-terminal region of an upstream enzyme and the N-terminal region of its downstream partner enzyme (48). These enzymes contain at least one module, and each module activates an amino acid that is incorporated into fengycin (28,30,41), allowing the activated amino acids to form a peptide on the enzyme chain (13). The modules typically contain an adenylation domain consisting of about 550 amino acids (10), which recognizes and adenylates an amino acid (13,42,44,51).…”