The linear pentadecapeptide gramicidin is assembled by four multimodular nonribosomal peptide synthetases that comprise 16 modules with 56 catalytic domains

Linear gramicidin is a membrane channel forming pentadecapeptide that is produced via the nonribosomal pathway. It consists of 15 hydrophobic amino acids with alternating l- and d-configuration forming a beta-helix-like structure. It has an N-formylated valine and a C-terminal ethanolamine. Here we report cloning and sequencing of the entire biosynthetic gene cluster as well as initial biochemical analysis of a new reductase domain. The biosynthetic gene cluster was identified on two nonoverlapping fosmids and a 13-kilobase pair (kbp) interbridge fragment covering a region of 74 kbp. Four very large open reading frames, lgrA, lgrB, lgrC, and lgrD with 6.8, 15.5, 23.3, and 15.3 kbp, were identified and shown to encode nonribosomal peptide synthetases with two, four, six, and four modules, respectively. Within the 16 modules identified, seven epimerization domains in alternating positions were detected as well as a putative formylation domain fused to the first module LgrA and a putative reductase domain attached to the C-terminal module of LgrD. Analysis of the substrate specificity by phylogenetic studies using the residues of the substrate-binding pockets of all 16 adenylation domains revealed a good agreement of the substrate amino acids predicted with the sequence of linear gramicidin. Additional biochemical analysis of the three adenylation domains of modules 1, 2, and 3 confirmed the colinearity of this nonribosomal peptide synthetase assembly line. Module 16 was predicted to activate glycine, which would then, being the C-terminal residue of the peptide chain, be reduced by the adjacent reductase domain to give ethanolamine, thereby releasing the final product N-formyl-pentadecapeptide-ethanolamine. However, initial biochemical analysis of this reductase showed only a one-step reduction yielding the corresponding aldehyde in vitro.     

Dissecting and exploiting nonribosomal peptide synthetases

Over the past decade striking advances in microbialgenetics have propelled a revolution in our ability todeduce, analyze and manipulate the biosynthesis of struc-turally complex and biologically important families of na-ture products, one most notable cla…     

ATPase activity of non-ribosomal peptide synthetases

Adenylation domains of non-ribosomal peptide synthetases (NRPS) catalyse the formation of aminoacyl adenylates, and in addition synthesize mono- and dinucleoside polyphosphates. Here, we show that NRPS systems furthermore contain an ATPase activity in the range of up to 2 P(i)/min. The hydrolysis rate by apo-tyrocidine synthetase 1 (apo-TY1) is enhanced in the presence of non-cognate amino acid substrates, correlating well with their structural features and the diminishing adenylation efficiency. A comparative analysis of the functional relevance of an analogous sequence motif in P-type ATPases and adenylate kinases (AK) allowed a putative assignment of the invariant aspartate residue from the TGDLA(V)R(K) core sequence in NRPS as the Mg(2+) binding site. Less pronounced variations in ATPase activity are observed in domains with relaxed amino acid specificity of gramicidin S synthetase 2 (GS2) and delta-(L-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), known to produce a set of substitutional variants of the respective peptide product. These results disclose new perspectives about the mode of substrate selection by NRPS.     

Purification of peptide synthetases involved in pristinamycin I biosynthesis.

Several assays of pristinamycin I synthetases based on adenylate or thioester formation were developed. Purification to near homogeneity of these enzymatic activities from cell extracts of Streptomyces pristinaespiralis showed that three enzymes could activate all pristinamycin I precursors. SnbA, a 3-hydroxypicolinic acid: AMP ligase activating the first pristinamycin I residue, was purified 200-fold, using an ATP-pyrophosphate exchange assay. This enzyme was shown to be a monomer with an Mr of 67,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then a multifunctional enzyme, consisting of two identical subunits (SnbC) with Mrs of 240,000 and able to bind covalently L-threonine as a thioester, was purified 100-fold. This protein also activated L-aminobutyric acid, which is further epimerized to generate the third residue of the pristinamycin I macrocycle. A third protein, consisting of two identical subunits (SnbD) with Mrs estimated to be between 250,000 and 350,000, was purified 200-fold. This large enzyme catalyzed thioesterification and subsequent N-methylation of 4-dimethylamino-L-phenylalanine, the fifth pristinamycin I residue. SnbD could also activate L-proline, the fourth pristinamycin I residue, and some preparations retained a low but significant activity for the last two pristinamycin I precursors. Finally, a single polypeptide chain (SnbE) with an Mr of 170,000, catalyzing L-phenylglycine-dependent ATP-pyrophosphate exchange, was purified 3,000-fold and characterized. Stepwise Edman degradation of the entire polypeptides or some of their internal fragments provided amino acid sequences for the four isolated proteins. The purified SnbE protein was further shown to be a proteolytic fragment of SnbD.     

Biosynthesis of natural products on modular peptide synthetases

Microbial nonribosomally processed peptides represent a large class of natural products including numerous important pharmaceutical agents, as well as other representatives that play a prevalent role in pathogenicity of certain microorganisms [M. A. Marahiel, T. Stachelhaus, and H. D. Mootz (1997). Chem. Rev. 97, 2651-2673]. Although diverse in structure, nonribosomally synthesized peptides have a common mode of biosynthesis. They are assembled on very large protein templates called peptide synthetases that exhibit a modular organization, allowing polymerization of monomers in an assembly-line-like mechanism.     

Genetic manipulation of non-ribosomal peptide synthetases to generate novel bioactive peptide products

Non-ribosomal peptide synthetases (NRPS) are large modular enzymes that govern the synthesis of numerous biotechnologically relevant products. Their mode of action is frequently compared to an assembly line, in which each module acts in a semi-autonomous but coordinated manner to add a specific monomer to a growing peptide chain, unfettered by ribosomal constraints. The modular nature of these systems offers tantalising prospects for synthetic biology, wherein the assembly line is re-engineered at a genetic level to generate a specific or combinatorial modified product. However, despite some success stories, a “one size fits all” approach to NRPS synthetic biology remains elusive. This review examines both rational and random mutagenesis strategies that have been employed to modify NRPS function, in an attempt to highlight key points that should be considered when seeking to re-engineer an NRPS biosynthetic template.     

Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases

The C-terminal thioesterase (TE) domains from nonribosomal peptide synthetases (NRPSs) catalyze the final step in the biosynthesis of diverse biologically active molecules. In many systems, the thioesterase domain is involved in macrocyclization of a linear precursor presented as an acyl-S-enzyme intermediate. The excised thioesterase domain from the tyrocidine NRPS has been shown to catalyze the cyclization of a peptide thioester substrate which mimics its natural acyl-S-enzyme substrate. In this work we explore the generality of cyclization catalyzed by isolated TE domains. Using synthetic peptide thioester substrates from 6 to 14 residues in length, we show that the excised TE domain from the tyrocidine NRPS can be used to generate an array of sizes of cyclic peptides with comparable kinetic efficiency. We also studied the excised TE domains from the NRPSs which biosynthesize the symmetric cyclic decapeptide gramicidin S and the cyclic lipoheptapeptide surfactin A. Both TE domains exhibit expected cyclization activity: the TE domain from the gramicidin S NRPS catalyzes head-to-tail cyclization of a decapeptide thioester to form gramicidin S, and the TE domain from the surfactin NRPS catalyzes stereospecific cyclization to form a macrolactone analogue of surfactin. With an eye toward generating libraries of cyclic molecules by TE catalysis, we report the solid-phase synthesis and TE-mediated cyclization of a small pool of linear peptide thioesters. These studies provide evidence for the general utility of TE catalysis as a means to synthesize a wide range of macrocyclic compounds.     

Carrier protein recognition in siderophore-producing nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) use phosphopantetheine (pPant) bearing carrier proteins to chaperone activated aminoacyl and peptidyl intermediates to the various enzymes that effect peptide synthesis. Using components from siderophore NRPSs that synthesize vibriobactin, enterobactin, yersiniabactin, pyochelin, and anguibactin, we examined the nature of the interaction of such cofactor-carrier proteins with acyl-activating adenylation (A) domains. While VibE, EntE, and PchD were all able to utilize "carrier protein-free" pPant derivatives, the pattern of usage indicated diversity in the binding mechanism, and even the best substrates were down at least 3 log units relative to the native cofactor-carrier protein. When tested with four noncognate carrier proteins, EntE and VibE differed both in the range of substrate utilization efficiency and in the distribution of the efficiencies across this range. Correlating sequence alignments to kinetic efficiency allowed for the construction of eight point mutants of VibE's worst substrate, HMWP2 ArCP, to the corresponding residue in its native VibB. Mutants S49D and H66E combined to increase activity 6.2-fold and had similar enhancing effects on the downstream condensation domain VibH, indicating that the two NRPS enzymes share carrier protein recognition determinants. Similar mutations of HMWP2 ArCP toward EntB had little effect on EntE, suggesting that the position of recognition determinants varies across NRPS systems.     

Modular structure of genes encoding multifunctional peptide synthetases required for non-ribosomal peptide synthesis

Abstract Peptide synthetases are large multienzyme complexes that catalyze the non-ribosomal synthesis of a structurally diverse family of bioactive peptides. They possess a multidomain structure and employ the thiotemplate mechanism to activate, modify and link together by amide or ester bonds the constituent amino acids of the peptide product. The domains, which represent the functional building units of peptide synthetases, appear to act as independent enzymes whose specific linkage order forms the protein-template that defines the sequence of the incorporated amino acids. Two types of domains have been characterized in peptide synthetases of bacterial and fungal origin: type I comprises about 600 amino acids and contains at least two modules involved in substrate recognition, adenylation and thioester formation, whereas type II domains carry in addition an insertion of about 430 amino acids that may function as a N-methyltransferase module. The role of other genes associated with bacterial opérons encoding peptide synthetases is also discussed.     

Assembly of aryl-capped siderophores by modular peptide synthetases and polyketide synthases

Bacterial siderophores assist pathogens in iron acquisition inside their hosts. They are often essential for achieving a successful infection, and their biosynthesis represents an attractive antibiotic target. Recently, several siderophore biosynthetic loci have been identified, and in vitro studies have advanced our knowledge of the biosynthesis of aryl-capped peptide and peptide–polyketide siderophores from Mycobacterium spp., Pseudomonas spp., Yersinia spp. and other bacteria. These studies also provided insights into the assembly of related siderophores and many secondary metabolites of medical relevance. Assembly of aryl-capped peptide and peptide–polyketide siderophores involves non-ribosomal peptide synthetase, polyketide synthase and non-ribosomal-peptide polyketide hybrid subunits. Analysis of these subunits suggests that their domains and modules are functionally and structurally independent. It appears that nature has selected a set of functional domains and modules that can be rearranged in different order and combinations to biosynthesize different products. Although much remains to be learned about modular synthetases and synthases, it is already possible to conceive strategies to engineer these enzymes to generate novel products.     

Phylogenetic analysis of condensation domains in the nonribosomal peptide synthetases

Condensation (C) domains in the nonribosomal peptide synthetases are capable of catalyzing peptide bond formation between two consecutively bound various amino acids. C-domains coincide in frequency with the number of peptide bonds in the product peptide. In this study, a phylogenetic approach was used to investigate structural diversity of bacterial C-domains. Phylogenetic trees show that the C-domains are clustered into three functional groups according to the types of substrate donor molecules. They are l-peptidyl donors, d-peptidyl donors, and N-acyl donors. The fact that C-domain structure is not subject to optical configuration of amino acid acceptor molecules supports an idea that the conversion from l to d-form of incorporating amino acid acceptor occurs during or after peptide bond formation. l-peptidyl donors and d-peptidyl donors are suggested to separate before separating the lineage of Gram-positive and Gram-negative bacteria in the evolution process.     

Design, construction and application of a fully automated equimolar peptide mixture synthesizer.

A fully automated peptide synthesizer has been constructed that is capable of the synthesis of equimolar peptide mixtures and the simultaneous synthesis of 36 individual peptides. The synthesizer was constructed from a workstation of our own design utilizing a Zymark robot arm. A Macintosh II computer coordinates the movements of the robotic arm, the switching of over 40 solenoid valves and the monitoring of sensors in the workstation. The robot hands are used to deliver solvents from pressurized spigot lines and to pipet amino acid solutions from reservoirs to an array of reaction vessels. Liquid dispensing, reagent mixing and solvent removal are controlled from a multifunction I/O board in the computer. The design features of the synthesizer are presented, as well as the characterization of multiple individual peptides, a simple mixture of 19 components, and a complex mixture of 15,625 components.     

Actinomycin synthetases. Multifunctional enzymes responsible for the synthesis of the peptide chains of actinomycin

Two enzymes were purified from actinomycin-synthesizing Streptomyces chrysomallus which could be identified as peptide synthetases involved in the biosynthesis of actinomycin. Actinomycin synthetase II activates the first two amino acids of the peptide chains of the peptide lactone antibiotic, threonine and valine (or isoleucine), as thioesters via their corresponding adenylates. It is a single polypeptide chain of Mr 225,000. Similarly, actinomycin synthetase III activates proline, glycine, and valine (the remaining three amino acids in the antibiotic) as thioesters and is a single polypeptide chain of about Mr 280,000. It also carries the methyltransferase function(s) for N-methylation of thioesterified glycine and valine. In addition, it catalyzes the formation of cyclo(sarcosyl-N-methyl-L-valine) from glycine, L-valine, and S-adenosyl-L-methionine at the expense of ATP. Although the cell-free synthesis of the peptide lactone was not as yet accomplished, the data provide evidence that together with the 4-methyl-3-hydroxyanthranilic acid-activating enzyme (now designated as actinomycin synthetase I) all amino acid-activating protein components of the actinomycin-synthesizing enzyme complex are identified.     

Single sequence of a helix-loop peptide confers functional anticodon recognition on two tRNA synthetases.

The specific aminoacylation of RNA oligonucleotides whose sequences are based on the acceptor stems of tRNAs can be viewed as an operational RNA code for amino acids that may be related to the development of the genetic code. Many synthetases also have direct interactions with tRNA anticodon triplets and, in some cases, these interactions are thought to be essential for aminoacylation specificity. In these instances, an unresolved question is whether interactions with parts of the tRNA outside of the anticodon are sufficient for decoding genetic information. Escherichia coli isoleucyl- and methionyl-tRNA synthetases are closely related enzymes that interact with their respective anticodons. We used binary combinatorial mutagenesis of a 10 amino acid anticodon binding peptide in these two enzymes to identify composite sequences that would confer function to both enzymes despite their recognizing different anticodons. A single peptide was found that confers function to both enzymes in vivo and in vitro. Thus, even in enzymes where anticodon interactions are normally important for distinguishing one tRNA from another, these interactions can be 'neutralized' without losing specificity of amino-acylation. We suggest that acceptor helix interactions may play a role in providing the needed specificity.     

Insulin-binding peptide. Design and characterization

The design and characterization of a six-amino acid-containing peptide that binds insulin is described. The amino acid sequence of the insulin-binding peptide (IBP) was determined from the strand of DNA complementary to the strand of DNA coding for the insulin molecule in the domain of the insulin monomer believed to interact with the insulin receptor. The IBP (Cys-Val-Glu-Glu-Ala-Ser) binds specifically to insulin in a saturable manner with a Kd of 3 nM. This binding process is time dependent and slightly temperature dependent, and the peptide appears to interact with insulin near the carboxyl terminus of the B-chain of insulin. Incubation of insulin with the peptide decreases insulin binding to the insulin receptor by 50%, with no effect on the affinity of insulin for the receptor and no effect on cellular insulin-stimulated deoxyglucose uptake. A polyclonal antibody produced against the IBP will inhibit specific insulin binding to intact cells by approximately 50%, with no effects on insulin-stimulated glucose uptake. From this data, we suggest that there are at least two domains of the insulin molecule through which it interacts with its receptor, the "binding region" of insulin, which is the domain blocked by the IBP, and the "message region" of insulin, through which insulin not only binds to the receptor, but also generates the cellular signal.     

Pseudomonas syringae phytotoxins: mode of action, regulation, and biosynthesis by peptide and polyketide synthetases.

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents.     

Analysis of engineered multifunctional peptide synthetases. Enzymatic characterization of surfactin synthetase domains in hybrid bimodular systems.

The combinatorial reorganization of distinct modules of multimodular peptide synthetases is of increasing interest for the generation of new peptides with optimized bioactive properties. Each module is at least composed of enzymatic domains responsible for the adenylation, thioester formation, and condensation of an amino acid residue of the final peptide product. We analyzed various possible fusion sites for the recombination of peptide synthetases and evaluated the impact of different recombination strategies on the amino acid adenylation and acyl-thioester formation activities of peptide synthetase modules. Hybrid bimodular peptide synthetases were generated by recombination of the corresponding reading frames encoding for L-glutamic acid- and L-leucine-specific modules of surfactin synthetase SrfA-A at presumed inner- and intradomainic regions. We demonstrate that fusions at a previously postulated hinge region, dividing the amino acid adenylating domains of peptide synthetase modules into two subdomains, and at the highly conserved 4'-phosphopantetheine binding motif in acyl-thioester forming domains resulted in enzymatically active hybrid domains. By contrast, most manipulations in condensation domains like deletions, the complete exchange or the construction of chimeric domains considerably reduced or completely abolished the amino acid adenylation and thioester formation activity of the hybrid module.     

Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics

Recently, the solved crystal structure of a phenylalanine-activating adenylation (A) domain enlightened the structural basis for the specific recognition of the cognate substrate amino acid in nonribosomal peptide synthetases (NRPSs). By adding sequence comparisons and homology modeling, we successfully used this information to decipher the selectivity-conferring code of NRPSs. Each codon combines the 10 amino residues of a NRPS A domain that are presumed to build up the substrate-binding pocket. In this study, the deciphered code was exploited for the first time to rationally alter the substrate specificity of whole NRPS modules in vitro and in vivo. First, the single-residue Lys239 of the L-Glu-activating initiation module C-A(Glu)-PCP of the surfactin synthetase A was mutated to Gln239 to achieve a perfect match to the postulated L-Gln-activating binding pocket. Biochemical characterization of the mutant protein C-A(Glu)-PCP(Lys239 --> Gln) revealed the postulated alteration in substrate specificity from L-Glu to L-Gln without decrease in catalytic efficiency. Second, according to the selectivity-conferring code, the binding pockets of L-Asp and L-Asn-activating A domains differs in three positions: Val299 versus Ile, His322 versus Glu, and Ile330 versus Val, respectively. Thus, the binding pocket of the recombinant A domain AspA, derived from the second module of the surfactin synthetases B, was stepwisely adapted for the recognition of L-Asn. Biochemical characterization of single, double, and triple mutants revealed that His322 represents a key position, whose mutation was sufficient to give rise to the intended selectivity-switch. Subsequently, the gene fragment encoding the single-mutant AspA(His322 --> Glu) was introduced back into the surfactin biosynthetic gene cluster. The resulting Bacillus subtilis strain was found to produce the expected so far unknown lipoheptapeptide [Asn(5)]surfactin. This indicates that site-directed mutagenesis, guided by the selectivity-conferring code of NRPS A domains, represents a powerful alternative for the genetic manipulation of NRPS biosynthetic templates and the rational design of novel peptide antibiotics.