General rules of fragmentation evidencing lasso structures in CID and ETD.

Lasso peptides constitute a structurally unique class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked structure in which the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Tandem mass spectrometry using collision induced dissociation (CID) and electron capture dissociation (ECD) have shown previously different fragmentation patterns for capistruin, microcin J25 and their corresponding branched-cyclic forms in which the C-terminal tail is unthreaded. In order to develop general rules that unambiguously discriminate the lasso and branched-cyclic topologies, this report presents experimental evidence for a set of twenty-one lasso peptides analyzed by CID and electron transfer dissociation (ETD). CID experiments on lasso peptides specifically yielded mechanically interlocked species with associated bi and yj fragments. For class II lasso peptides, these lasso-specific fragments were observed only for peptides in which the loop, located above the macrolactam ring, was strictly longer than four amino acid residues. For class I and III lasso peptides, part of the C-terminal tail remains covalently linked to the macrolactam ring by disulfide bonds; associated bi and yj fragments therefore do not clearly constitute a signature of the lasso topology. ETD experiments of lasso peptides showed a significant increase of hydrogen migration events in the loop region when compared to their branched-cyclic topoisomers, leading to the formation of specific ci˙/z'j fragments for all lasso peptides, regardless of their class and loop size. Our experiments enabled us to establish general rules for obtaining structural details from CID and ETD fragmentation patterns, obviating the need for structure determination by NMR or X-ray crystallography.

Rational design of peptide antibiotics by targeted replacement of bacterial and fungal domains.

Peptide synthetases involved in the nonribosomal synthesis of peptide secondary metabolites possess a highly conserved domain structure. The arrangement of these domains within the multifunctional enzymes determines the number and order of the amino acid constituents of the peptide product. A general approach has been developed for targeted substitution of amino acid-activating domains within the srfA operon, which encodes the protein templates for the synthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis. Exchange of domain-coding regions of bacterial and fungal origin led to the construction of hybrid genes that encoded peptide synthetases with altered amino acid specificities and the production of peptides with modified amino acid sequences.

A superfamily of proteins that contain the cold-shock domain.

Members of a family of cold-shock proteins (CSPs) are found throughout the eubacterial domain and appear to function as RNA-chaperones. They have been implicated in various cellular processes, including adaptation to low temperatures, cellular growth, nutrient stress and stationary phase. The discovery of a domain--the cold-shock domain--that shows strikingly high homology and similar RNA-binding properties to CSPs in a growing number of eukaryotic nucleic-acid-binding proteins suggests that these proteins have an ancient origin.

Exploring the domain structure of modular nonribosomal peptide synthetases.

Recently, considerable insight has been gained into the modular organization of nonribosomal peptide synthetases (NRPS). The three-dimensional structures of domains associated with substrate adenylation and covalent binding have been solved as well as the structure of a priming enzyme required for the post-translational modification of NRPS. Taken together, these studies will help us to understand the architecture of these mega-enzymes.

Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases.

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large modular enzymes responsible for the synthesis of a variety of microbial bioactive peptides. They consist of modules that each recognise and incorporate one specific amino acid into the peptide product. A module comprises several domains, which carry out the individual reaction steps. After activation by the adenylation domain, the amino acid substrate is covalently tethered to a 4'-phosphopantetheinyl cofactor of a peptidyl carrier domain (PCP) that passes the substrate to the reaction centres of the consecutive domains. RESULTS: The solution structure of PCP, a distinct peptidyl carrier protein derived from the equivalent domain of an NRPS, was solved using NMR techniques. PCP is a distorted four-helix bundle with an extended loop between the first two helices. Its overall fold resembles the topology of acyl carrier proteins (ACPs) from Escherichia coli fatty acid synthase and actinorhodin polyketide synthase from Streptomyces coelicolor; however, the surface polarity and the length and relative alignment of the helices are different. The conserved serine, which is the cofactor-binding site, has the same location as in the ACPs and is situated within a stretch of seven flexible residues. CONCLUSIONS: The structure of PCP reflects its character as a protein domain. The fold is well defined between residues 8 and 82 and the structural core of the PCP domain can now be defined as a region spanning 37 amino acids in both directions from the conserved serine. The flexibility of the post-translationally modified site might have implications for interactions with the cooperating proteins or NRPS domains.

Tn10 insertional mutations of Bacillus subtilis that block the biosynthesis of bacilysin.

Transposon mutagenesis was employed to isolate the gene(s) related with the biosynthesis of dipeptide antibiotic in Bacillus subtilis PY79 (a prototrophic derivative of the standard 168 strain). The blocked mutants were phenotypically selected from the transposon library by bioassay and the complete loss of biosynthetic ability was verified through ESI-mass spectrometry analysis. Four different bacilysin nonproducer mutants (Bac(-)::Tn10(ori-spc)) were isolated from the transposon library. The genes involved in bacilysin biosynthesis were identified as thyA (thymidilate synthetase), ybgG (unknown; similar to homocysteine methyl transferase) and oppA (oligopeptide permease), respectively. The other blocked gene was yvgW (unknown; similar to heavy metal-transporting ATPase); however, backcross studies did not verify its involvement in bacilysin biosynthesis. This gene, on the other hand, appeared to be necessary for efficient sporulation and transformation. Opp involvement was significant as it suggested that bacilysin biosynthesis is under or a component of the quorum sensing pathway which has been shown to be responsible for the establishment of sporulation, competence development and onset of surfactin biosynthesis. For verification, it was necessary to check the involvement of peptide pheromones (PhrA or PhrC) internalized by the Opp system and response regulator ComA as the essential components of this global control. phrA, phrC and comA deleted mutants of PY79 were thus constructed and the latter two genes were shown to be essential for bacilysin biosynthesis.

Molecular characterization of the transition state regulator AbrB from Bacillus stearothermophilus.

The Bacillus subtilis transition state regulator AbrB(su) is a DNA-binding protein that acts on several genes either as activator, repressor, or preventer. However, among genes under its control, neither common binding sites could be identified nor could the structural features of this broad and specific interaction be elucidated. Attempts to elucidate these interesting features by crystallizing AbrB(su) have failed so far. Therefore, to solve this problem, we focused in this work on identifying an AbrB(su) homologue from Bacillus stearothermophilus. Using a novel method, the entire abrB(st) gene of B. stearothermophilus was cloned and sequenced. The gene encodes a 95 amino acid protein that shows 77% identity and 85% similarity to the mesophilic B. subtilis protein. A calmodulin binding peptide-tagged fusion of the thermophilic gene was constructed for overexpression and efficient affinity column purification of the AbrB(st) protein. The purified protein showed, after removal of the tag, an oligomerization behavior through hexamer formation that is essential for its DNA binding activity.

Biosynthetic systems for nonribosomal peptide antibiotic assembly.

Modular peptide synthetases, which act as the protein templates for the synthesis of a large number of peptide antibiotics and siderophores, hold great potential for the development of novel compounds. Recently, significant progress has been made towards understanding their molecular architecture and substrate specificity. The first crystal structure of a peptide synthetase has been solved, and the enzymes responsible for post-translational modification of peptide synthetases have recently been discovered. These will allow addressing important yet poorly understood mechanistic aspects.

Protein templates for the biosynthesis of peptide antibiotics.

Peptide synthetases of microbial origin can act as protein templates for the biosynthesis of unusual, often pharmacologically active, peptides of diverse structure and biological activity. Specific repeated modules in the synthetases each contain at least two distinct domains, required for substrate adenylation and thiolation, that define the sequence and length of the peptide product. The first crystal structure of an adenylation domain has provided insights into the mechanism of substrate recognition and activation.

Dipeptide formation on engineered hybrid peptide synthetases.

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are modular 'megaenzymes' that catalyze the assembly of a large number of bioactive peptides using the multiple carrier thiotemplate mechanism. The modules comprise specific domains that act as distinct units to catalyze specific reactions associated with substrate activation, modification and condensation. Such an arrangement of biosynthetic templates has evoked interest in engineering novel NRPSs. RESULTS: We describe the design and construction of a set of dimodular hybrid NRPSs. By introducing domain fusions between adenylation and thiolation (PCP) domains we designed synthetic templates for dipeptide formation. The predicted dipeptides, as defined by the specificity and arrangement of the adenylation domains of the constructed templates, were synthesized in vitro. The effect of the intramolecular fusion was investigated by determining kinetic parameters for substrate adenylation and thiolation. The rate of dipeptide formation on the artificial NRPSs is similar to that of natural templates. CONCLUSIONS: Several new aspects concerning the tolerance of NRPSs to domain swaps can be deduced. By choosing the fusion site in the border region of adenylation and PCP domains we showed that the PCP domain exhibits no general substrate selectivity. There was no suggestion that selectivity of the condensation reaction was biased towards the donor amino acid, whereas at the acceptor position there was a size-determined selection. In addition, we demonstrated that a native elongation module can be converted to an initiation module for peptide-bond formation. These results represent the first example of rational de novo synthesis of small peptides on engineered NRPSs.

How do peptide synthetases generate structural diversity?

Many low-molecular-weight peptides of microbial origin are synthesized nonribosomally on large multifunctional proteins, termed peptide synthetases. These enzymes contain repeated building blocks in which several defined domains catalyze specific reactions of peptide synthesis. The order of these domains within the enzyme determines the sequence and structure of the peptide product.

Biochemical characterization of peptidyl carrier protein (PCP), the thiolation domain of multifunctional peptide synthetases.

BACKGROUND: A structurally diverse group of bioactive peptides is synthesized by peptide synthetases which act as templates for a growing peptide chain, attached to the enzyme via a thioester bond. The protein templates are composed of distinctive substrate-activating modules, whose order dictates the primary structure of the corresponding peptide product. Each module contains defined domains that catalyze adenylation, thioester and peptide bond formation, as well as substrate modifications. To show that a putative thiolation domain (PCP) is involved in covalent binding and transfer of amino acyl residues during non-ribosomal peptide synthesis, we have cloned and biochemically characterized that region of tyrocidine synthetase 1, TycA. RESULTS: The 327-bp gene fragment encoding PCP was cloned using its homology to the genes for the acyl carrier proteins of fatty acid and polyketide biosynthesis. The protein was expressed as a His6 fusion protein, and purified in a single step by affinity chromatography. Incorporation of beta-[3H]alanine, a precursor of coenzyme A, demonstrated the modification of PCP with the cofactor 4'-phosphopantetheine. When an adenylation domain is present to supply the amino adenylate moiety, PCP can be acylated in vitro. CONCLUSIONS: PCP can bind covalently to the cofactor phosphopantetheine and can subsequently be acylated, strongly supporting the multiple carrier model of non-ribosomal peptide synthesis. The adenylation and thiolation domains can each act as independent multifunctional enzymes, further confirming the modular structure of peptide synthases, and can also perform sequential steps in trans, as do multienzyme complexes.

Exploring the impact of different thioesterase domains for the design of hybrid peptide synthetases.

BACKGROUND: A large number of pharmacologically important peptides are synthesized by multifunctional enzymes, the nonribosomal peptide synthetases (NRPSs). The thioesterase (Te) domain at the C-terminus of the last NRPS catalyzes product cleavage by hydrolysis or complex macrocyclization. Recent studies with excised Te domains and peptidyl-S-N-acetyl cysteamine substrate substitutes led to substantial insights in terms of cyclization activity and substrate tolerance of these enzymes. Their use in engineered hybrid NRPSs is an interesting but yet only little explored target for approaches to achieve new structural diversity and designed products. RESULTS: To study the capability of various Te domains to function in hybrid NRPSs, six different Te domains that catalyze different modes of termination in their natural systems were fused to a bimodular model NRPS system, consisting of the first two modules of tyrocidine NRPS, TycA and ProCAT. All Te domains were active in hydrolyzing the enzymatically generated dipeptide substrate D-Phe-Abu from the NRPS template with, however, greatly varying turnover rates. Two Te domains were also capable of hydrolyzing the substrate D-Phe-Pro and partially cyclized the D-Phe-Abu dipeptide, indicating that in an artificial context Te domains may display hydrolytic and cyclization activities that are not easily predictable. CONCLUSIONS: Te domains from heterologous NRPSs can be utilized for the construction of hybrid NRPSs. This is the first comparative study to explore their influence on the product pattern. The inherent specificity and regioselectivity of Te domains should allow control of the desired product cleavage, but can also lead to other modes of termination potentially useful for generating structural diversity. Our results provide the first data for choosing the proper Te domain for a particular termination reaction.

The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases.

BACKGROUND: Many pharmacologically important peptides are synthesized nonribosomally by multimodular peptide synthetases (NRPSs). These enzyme templates consist of iterated modules that, in their number and organization, determine the primary structure of the corresponding peptide products. At the core of each module is an adenylation domain that recognizes the cognate substrate and activates it as its aminoacyl adenylate. Recently, the crystal structure of the phenylalanine-activating adenylation domain PheA was solved with phenylalanine and AMP, illustrating the structural basis for substrate recognition. RESULTS: By comparing the residues that line the phenylalanine-binding pocket in PheA with the corresponding moieties in other adenylation domains, general rules for deducing substrate specificity were developed. We tested these in silico 'rules' by mutating specificity-conferring residues within PheA. The substrate specificity of most mutants was altered or relaxed. Generalization of the selectivity determinants also allowed the targeted specificity switch of an aspartate-activating adenylation domain, the crystal structure of which has not yet been solved, by introducing a single mutation. CONCLUSIONS: In silico studies and structure-function mutagenesis have defined general rules for the structural basis of substrate recognition in adenylation domains of NRPSs. These rules can be used to rationally alter the specificity of adenylation domains and to predict from the primary sequence the specificity of biochemically uncharacterized adenylation domains. Such efforts could enhance the structural diversity of peptide antibiotics such as penicillins, cyclosporins and vancomycins by allowing synthesis of 'unnatural' natural products.

The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases.

BACKGROUND: The branched cyclic dodecylpeptide antibiotic bacitracin, produced by special strains of Bacillus, is synthesized nonribosomally by a large multienzyme complex composed of the three bacitracin synthetases BA1, BA2 and BA3. These enzymes activate and incorporate the constituent amino acids of bacitracin by a thiotemplate mechanism in a pathway driven by a protein template. The biochemical features of these enzymes have been studied intensively but little is known about the molecular organization of their genes. RESULTS: The entire bacitracin synthetase operon containing the genes bacA-bacC was cloned and sequenced, identifying a modular structure typical of peptide synthetases. The bacA gene product (BA1, 598kDa) contains five modules, with an internal epimerization domain attached to the fourth; bacB encodes BA2 (297kDa), and has two modules and a carboxy-terminal epimerization domain; bacC encodes BA3, five modules (723kDa) with additional internal epimerization domains attached to the second and fourth. A carboxy-terminal putative thioesterase domain was also detected in BA3. A putative cyclization domain was found in BA1 that may be involved in thiazoline ring formation. The adenylation/thioester-binding domains of the first two BA1 modules were overproduced and the detected amino-acid specificity coincides with the first two amino acids in bacitracin. Disruption of chromosomal bacB resulted in a bacitracin-deficient mutant. CONCLUSIONS: The genes encoding the bacitracin synthetases BA1, BA2 and BA3 are organized in an operon, the structure of which reflects the modular architecture expected of peptide synthetases. In addition, a putative thiazoline ring formation domain was identified in the BA1 gene.

A new enzyme superfamily - the phosphopantetheinyl transferases.

BACKGROUND: All polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases require posttranslational modification of their constituent acyl carrier protein domain(s) to become catalytically active. The inactive apoproteins are converted to their active holo-forms by posttranslational transfer of the 4'-phosphopantetheinyl (P-pant) moiety of coenzyme A to the sidechain hydroxyl of a conserved serine residue in each acyl carrier protein domain. The first P-pant transferase to be cloned and characterized was the recently reported Escherichia coli enzyme ACPS, responsible for apo to holo conversion of fatty acid synthase. Surprisingly, initial searches of sequence databases did not reveal any proteins with significant peptide sequence similarity with ACPS. RESULTS: Through refinement of sequence alignments that indicated low level similarity with the ACPS peptide sequence, we identified two consensus motifs shared among several potential ACPS homologs. This has led to the identification of a large family of proteins having 12-22 % similarity with ACPS, which are putative P-pant transferases. Three of these proteins, E. coli EntD and o195, and B. subtilis Sfp, have been overproduced, purified and found to have P-pant transferase activity, confirming that the observed low level of sequence homology correctly predicted catalytic function. Three P-pant transferases are now known to be present in E. coli (ACPS, EntD and o195); ACPS and EntD are specific for the activation of fatty acid synthase and enterobactin synthetase, respectively. The apo-protein substrate for o195 has not yet been identified. Sfp is responsible for the activation of the surfactin synthetase. CONCLUSIONS: The specificity of ACPS and EntD for distinct P-pant-requiring enzymes suggests that each P-pant-requiring synthase has its own partner enzyme responsible for apo to holo activation of its acyl carrier domains. This is the first direct evidence that in organisms containing multiple P-pant-requiring pathways, each pathway has its own posttranslational modifying activity.

Design and application of multimodular peptide synthetases.

Nonribosomal peptide synthetases produce bioactive peptides of great structural diversity. Their modular organization makes them amenable to the construction of hybrid enzymes that synthesize novel products. New strategies for combinatorial approaches are being developed from the recent advances in nonribosomal peptide synthesis on the genetic, biochemical and structural level.

Effect of pH and phosphate ions on self-association properties of the major cold-shock protein from Bacillus subtilis.

The intermolecular interactions of the major cold-shock protein from Bacillus subtilis (CspB) in solution in the presence of different salts, including phosphate, have been studied by means of scanning calorimetry and size-exclusion chromatography. Calorimetric results indicate that, in all cases, protein unfolding can be approximated by a 2-state model, but the modes of unfolding can differ depending on the conditions. In the presence of phosphate, the cooperative folding unit is a monomer, whereas in the absence of phosphate, the cooperative unit is a dimer. The difference in the self-association of CspB in the presence and absence of phosphate was supported by size-exclusion chromatography. These results are compared with recent structural studies of CspB in crystal and in solution.

Mapping of the Bacillus subtilis cspB gene and cloning of its homologs in thermophilic, mesophilic and psychrotrophic bacilli.

The Bacillus subtilis cold shock (CS)-inducible gene, cspB, encoding the nucleic-acid-binding, major CS protein CspB, is located at about 80 degrees on the B. subtilis genetic map. Using this cspB as a probe, the CspB-encoding genes from two thermophilic bacilli were cloned and characterized. The nucleotide (nt) sequences of the B. caldolyticus and B. stearothermophilus cspB coding regions are 78 and 76% identical to the B. subtilis cspB and the deduced amino acid (aa) sequences revealed 84 and 82% identity, respectively. The cspB genes of the mesophilic B. globigii and the some what psychrotrophic B. globisporus, were amplified by PCR using mixed degenerate oligodeoxyribonts based on the 5' and 3' ends of B. subtilis cspB. The nt sequence comparisons of the resulting cloned PCR fragments revealed 98 to 99% identity to cspB of B. subtilis and 97% aa identity to the CspB protein. The high conservation of CspB within the genus Bacillus and the presence of a related nucleic acid-binding domain within several eukaryotic transcription factors implies an important common biological function that seems to be highly conserved from bacteria to man.

Multidomain enzymes involved in peptide synthesis.

Biosynthesis of peptides in non-ribosomal systems is catalyzed by multifunctional enzymes that employ the thio-template mechanism. Recent studies on the analysis of the primary structure of several peptide synthetases have revealed that they are organized in highly conserved and repeated functional domains. The aligned domains provide the template for peptide synthesis, and their order determines the sequence of the peptide product.