Substrate Specificity of Hybrid Modules from Peptide Synthetases

Abstract

Homologous modules from two different peptide synthetases were analyzed for functionally equivalent regions. Hybrids between the coding regions of the phenylalanine-activating module of tyrocidine synthetase and the valine-activating module of surfactin synthetase were constructed by combining the two reading frames at various highly conserved consensus sequences. The resulting DNA fragments were expressed in Escherichia coli as C-terminal fusions to the gene encoding for the maltose-binding protein. The fusion proteins were purified, and the amino acid specificities, the acceptance of different nucleotide analogues, and the substrate binding affinities were analyzed. We found evidence for a large N-terminal domain and a short C-terminal domain of about 19 kDa within the two modules, which are separated by the sequence motif GELCIGG. The two domains could be reciprocally transferred between the two modules, and the constructed hybrid proteins showed amino acid adenylating activity. Hybrid proteins fused at various consensus motifs within the two domains were inactive, indicating that the domains may fold independently and represent complex functional units. The N-terminal domain was found to be responsible for the amino acid specificity of the modules, and it is also involved in the recognition of the ribosyl and the phosphate moieties of the nucleotide substrate. For tyrocidine synthetase I, we could confine the sites for amino acid specificity to a region of 330 residues. The C-terminal domain is essential for the enzymatic activity and has a strong impact on the specific activity of the modules.Tyrocidine synthetase and surfactin synthetase are multifunctional peptide synthetases produced by Bacillus brevis and Bacillus subtilis, respectively (1-5). The enzymes belong to a superfamily of adenylate-forming enzymes (6 -8). Small peptides like tyrocidine A and surfactin are formed by a nonribosomal pathway according to the thio-template mechanism (6, 9). Prior to incorporation, amino acids and related compounds are activated as adenylates by cleavage of ATP and release of pyrophosphate. Peptide synthetases exhibit a modular structure with several linked modules of about 100 kDa (1, 10 -16). Each module is responsible for recognition, activation, and incorporation of a specific amino acid constituent into the peptide product. Various modules of peptide synthetases have been sequenced, and several highly conserved motifs were found (7,8,11,14,(17)(18)(19). Mutagenesis and cross-linking experiments gave evidence for the involvement of most of these motifs in the binding of ATP and the adenylate forming activity (20 -23). A serine residue in the highly conserved sequence motif LGG(H/D)S at the C terminus of the modules was clearly identified as the site for covalent attachment of a phosphopantetheine cofactor (24 -26). The deletion of the cofactor attachment site in the phenylalanine-activating modules of tyrocidine synthetase I and gramicidin S synthetase I did not affect the amino acid adenylate forming activity (...

The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains

“…They are comparable to those determined for wild-type GrsB, the most closely related peptide synthetase (18). The K m values obtained are also in the same order of magnitude as those reported for other peptide synthetases (8,33,45) and for aminoacyl-tRNA synthetases. The PheA4 and TyrA7 domains did indeed have higher affinities to L-Trp (K m values of 0.08 and 0.10 mM, respectively) than to L-Phe (0.45 mM) and L-Tyr (0.30 mM), respectively.…”

“…Although heterologous expression of peptide synthetase fragments has previously been described by a few authors, in those cases, larger proteins corresponding mostly to an entire module (110 to 140 kDa) were examined (8,14,16,23,24). C-terminal-truncated GrsA and TycA derivatives are the only reported examples of recombinant minimal adenylation domains expressed in a heterologous host, but they retained their native N termini and were constructed from relatively simple peptide synthetases (comprising only one module) (6,45).…”

Analysis of Engineered Multifunctional Peptide Synthetases

“…The resulting plasmids were named pH-SrfA-A, pH-SrfA-C, and pH-SrfM1, and the proteins were synthesized with a terminal poly(His) 6 -tag. The valine activating module Srf-M4 was encoded from plasmid pH-SrfM4 as described previously (20). The two modules Srf-M1 and Srf-M4 carry intact C-and A-domains, and were deleted for their T-domains.…”

Genetic analysis of the biosynthesis of non-ribosomal peptide- and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1/3

“…The core of each module is the amino acid adenylation domain characterized by the motifs SGTTGxPKG and TGD. These are believed to function in ATP binding and hydrolysis (Elsner et al 1997;Marahiel et al 1997). The peptidyl (PCP) and the acyl carrier protein (ACP) domains contain the highly conserved sequence motif [I/L]GG[D/H]SL at their thiolation sites; the invariant serine binds the 4¢-phosphopantotheine cofactor (Leenders et al 1998;Lambalot et al 1996).…”