Nonribosomal Peptide Synthetase Assembly Lines Research Articles

Transient Domain Interactions in Non-Ribosomal Peptide Synthetases

Non-ribosomal peptide synthetases (NRPSs) are multi-module, multi-domain enzymes, synthesizing a wealth of important natural products in bacteria and fungi. These products often find pharmaceutical applications as antibiotics, antitumor agents or immunosuppressant drugs. NRPS use a remarkable assembly line organization to covalently load substrates into each module and catalyze peptide bond formation between substrates loaded on adjacent modules. The substrates may optionally be modified by tailoring domains. These multiple catalytic steps require a series of sequential domain interactions, which are currently poorly understood. We have used NMR to study structures and dynamics of NRPS single- and multi-domains. We show that the domains are neither subject to random interactions nor do they display a fixed quaternary organization. Instead, NRPSs are subject to multiple transient domain interactions which are accompanied by a modulation of their internal dynamics. Understanding the dynamic mechanism of NRPS domain communication may open the venue to efficient NRPS assembly line reprogramming and the production of novel pharmaceuticals.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Working outside the protein‐synthesis rules: insights into non‐ribosomal peptide synthesis

Non-ribosomally synthesized microbial peptides show remarkable structural diversity and constitute a widespread class of the most potent antibiotics and other important pharmaceuticals that range from penicillin to the immunosuppressant cyclosporine. They are assembled independent of the ribosome in a nucleic acids-independent way by a group of multimodular megaenzymes called non-ribosomal peptide synthetases. These biosynthetic machineries rely not only on the 20 canonical amino acids, but also use several different building blocks, including D-configured- and beta-amino acids, methylated, glycosylated and phosphorylated residues, heterocyclic elements and even fatty acid building blocks. This structural diversity leads to a high density of functional groups, which are often essential for the bioactivity. Recent biochemical and structural studies on several non-ribosomal peptide synthetase assembly lines have substantially contributed to the understanding of the molecular mechanisms and dynamics of individual catalytic domains underlying substrate recognition and substrate shuffling among the different active sites as well as peptide bond formation and the regio- and stereoselective product release.

The Biosynthesis of Teicoplanin-Type Glycopeptide Antibiotics: Assignment of P450 Mono-Oxygenases to Side Chain Cyclizations of Glycopeptide A47934

Streptomyces toyocaensis produces A47934, a teicoplanin-like type-IV glycopeptide with antibiotic activity against methicillin-resistant Staphylococcus aureus. A47934 differs from the type-I vancomycin glycopeptides, which possess a tricyclic peptide backbone, by the presence of an additional ring closure between the aromatic amino acids 1 and 3. To elucidate the order of crosslinking reactions, P450 mono-oxygenase-inactivation mutants (DeltastaF, DeltastaG, DeltastaH, and DeltastaJ) of the A47934 producer were generated, and the accumulated intermediates were analyzed. Thus, the formation of each crosslink could unambiguously be assigned to a specific oxygenase. The structure of the released intermediates from the wild-type nonribosomal peptide synthetase assembly line facilitated the determination of the cyclization order. Unexpectedly, the additional ring closure in A47934, catalyzed by StaG, is the second oxygenase reaction.

Generation of D Amino Acid Residues in Assembly of Arthrofactin by Dual Condensation/Epimerization Domains

The first 6 residues of the biosurfactant lipopeptidolactone arthrofactin have the D configuration, yet none of the 11 modules of the nonribosomal peptide synthetase assembly line have epimerization domains. We show that the two-module ArfA subunit and the first module of the ArfB subunit, which act in tandem to produce the N-acyl-D-Leu1-D-Asp2-D-Thr3-S-protein intermediate, activate the L amino acids and epimerize them as the aminoacyl-S-pantetheinyl T domain intermediates before the next downstream condensation. The condensation (C) domains are shown to have (D)C(L) chirality in peptide bond formation. The upstream aminoacyl/peptidyl moiety is epimerized before condensation only when the condensation domains are simultaneously presented with the L-aminoacyl-S-pantetheinyl acceptor. These (D)C(L) catalysts are dual function condensation/epimerization domains that can be predicted by bioinformatics analysis to be responsible for incorporation of all D residues in arthrofactin and of D residues in syringomycin, syringopeptin, and ramoplanin synthetases.

In silico analysis of the adenylation domains of the freestanding enzymes belonging to the eucaryotic nonribosomal peptide synthetase‐like family

This work presents a computational analysis of the molecular characteristics shared by the adenylation domains from traditional nonribosomal peptide synthetases (NRPSs) and the group of the freestanding homologous enzymes: alpha-aminoadipate semialdehyde dehydrogenase, alpha-aminoadipate reductase and the protein Ebony. The results of systematic sequence comparisons allow us to conclude that a specificity-conferring code, similar to that described for the NRPSs, can be recognized in such enzymes. The structural and functional roles of the residues involved in the substrate selection and binding are proposed through the analysis of the predicted interactions of the model active sites and their respective substrates. The indications deriving from this study can be useful for the programming of experiments aimed at a better characterization and at the engineering of this emerging group of single NRPS modules that are responsible for amino acid selection, activation and modification in the absence of other NRPS assembly line components.

Macrolactamization of Glycosylated Peptide Thioesters by the Thioesterase Domain of Tyrocidine Synthetase

The 35 kDa thioesterase (TE) domain excised from the megadalton tyrocidine synthetase (Tyc Syn) retains autonomous capacity to macrocyclize peptidyl thioesters to D-Phe1-L-Leu10-macrolactams. Since a number of nonribosomal peptides undergo O-glycosylation events during tailoring to gain biological activity, the Tyc Syn TE domain was evaluated for cyclization capacity with glycosylated peptidyl-S-NAC substrates. First, Tyr7 was replaced with Tyr(beta-D-Gal) and Tyr(beta-D-Glc) as well as with Ser-containing beta-linked D-Gal, D-Glc, D-GlcNAc, and D-GlcNH2, and these new analogs were shown to be cyclized with comparable kcat/Km catalytic efficiency. Similarly, Gal- or tetra-O-acetyl-Gal-Ser could also be substituted at residues 5, 6, and 8 in the linear decapeptidyl-S-NAC sequences and cyclized without substantial loss in catalytic efficiency by Tyc Syn TE. The cyclic glycopeptides retained antibiotic activity as membrane perturbants in MIC assays, opening the possibility for library construction of cyclic glycopeptides by enzymatic macrocyclization.

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.

Epimerization of an L-cysteinyl to a D-cysteinyl residue during thiazoline ring formation in siderophore chain elongation by pyochelin synthetase from Pseudomonas aeruginosa.

The thiazoline-containing siderophores pyochelin, yersiniabactin, and Micacocidin A all have D-thiazoline rings, participating in high-affinity chelation of ferric iron. However, studies with pyochelin (Pch) synthetase and yersiniabactin (Ybt) synthetase reconstituted from pure protein components have shown that only L-cysteine is activated and tethered as a covalent aminoacyl-S-enzyme intermediate. Nor are any of the canonical epimerase domains of nonribosomal peptide synthetase (NRPS) assembly lines found in the Ybt or Pch synthetase modules. Here, we report that the PchE subunit of the Pch synthetase exchanges solvent deuterium into the C(2) center of the thiazoline moieties during siderophore chain elongation. Both PchE and HMWP2, from Ybt synthetase, subunits have a 310-360-residue insert in their amino acid activation domains that look like defective methyltransferase (MT) domains. We suggest these inserts are noncanonical epimerase domains, reversibly deprotonating and reprotonating acyl-S-enzyme intermediates at the C(2) locus. The PchE subunit does not epimerize the Cys-S-enzyme intermediate, but once amide bond formation from a benzoyl-S-PchE donor is catalyzed by the cyclization (Cy) domain of PchE, the N-benzoyl-Cys-S-PchE intermediate is present as a D,L-mixture. The subsequent phenylthiazolinyl-S-PchE intermediate, arising from cyclodehydration of the N-benzoyl-Cys-S-PchE intermediate, is likewise a D,L-mixture on hydrolytic release and enantiomer analysis. These results suggest a default role for MT domains of NRPS assembly lines in generating alpha-carbanionic species from thioester intermediates during siderophore chain elongation.

BlmIII and BlmIV nonribosomal peptide synthetase-catalyzed biosynthesis of the bleomycin bithiazole moiety involving both in cis and in trans aminoacylation.

Cloning and sequence analysis of the bleomycin (BLM) biosynthetic gene cluster predicted that the two nonribosomal peptide synthetases (NRPSs), BlmIV and BlmIII, are responsible for the biosynthesis of the BLM bithiazole moiety. BlmIV is a seven domain (C(2)-A(2)-PCP(2)-Cy(1)-A(1)-PCP(1)-Cy(0)) NRPS, and BlmIII is a three domain (A(0)-PCP(0)-Ox) NRPS. The three domains of Cy(1)-A(1)-PCP(1) residing on the BlmIV subunit, the four domains of Cy(0) residing on the BlmIV subunit, and A(0)-PCP(0)-Ox residing on the BlmIII subunit constitute the two thiazole-forming NRPS-1 and NRPS-0 modules, respectively. BlmIII-A(0) was predicted to be nonfunctional, raising the question of how the NRPS-0 module activates and loads the Cys substrate to its cognate BlmIII-PCP(0). The NRPS-0 module consists of domains residing on two different subunits, requiring precise protein-protein interaction. Here, we report the production of the BlmIV and BlmIII NRPSs as an excised domain(s), module, or intact subunit form and biochemical characterizations of the resultant enzymes in vitro for their roles in BLM bithiazole biosynthesis. Our results (a) confirm that BlmIII-A(0) is a naturally occurring nonfunctional mutant, (b) demonstrate that BlmIV-A(1) activates Cys and catalyzes both in cis aminoacylation of BlmIV-PCP(1) (for NRPS-1) and in trans aminoacylation of BlmIII-PCP(0) (for NRPS-0), and (c) reveal that the C-terminus of the BlmIV subunit, characterized by the unprecedented AGHDDD(G) and PGHDDG repeats, is absolutely required for in trans aminoacylation of BlmIII-PCP(0). These findings underscore the flexibility and versatility of NRPSs in both structure and mechanism for natural product biosynthesis and provide an outstanding opportunity to study the molecular recognition and protein-protein interaction mechanism in NRPS assembly line enzymology.

Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis.

Nonribosomal peptide synthetases (NRPSs) make many natural products of clinical importance, but a deeper understanding of the protein domains that compose NRPS assembly lines is required before these megasynthetases can be effectively engineered to produce novel drugs. The N-terminal amide bond-forming condensation (C) domain of the enterobactin NRPS EntF was excised from the multidomain synthetase using endpoints determined from sequence alignments and secondary structure predictions. The isolated domain was well-folded when compared by circular dichroism to the vibriobactin NRPS VibH, a naturally free-standing C domain. The EntF domain was also fully functional in an assay based on a synthetic small-molecule substrate, seryl N-acetylcysteamine. Active site mutants of the EntF C domain were surprisingly inactive in vitro as compared to their VibH counterparts, yet maintained the overall domain structure. An in vivo assay was developed in the context of the full-length EntF protein to more sensitively probe the activity level of the C domain mutants, and this supported strong effects for the active site mutations. The crucial role of histidine-138 was confirmed by assay of the full-length protein in vitro. These results suggest a strong resemblance of catalysis by the EntF C domain to chloramphenicol acetyltransferase, including an active site organized by an arginine-aspartate salt bridge, a key histidine acting as a general base, and an asparagine instead of a serine stabilizing the proposed tetrahedral intermediate by hydrogen bonding. The precise definition of a functional C domain excised from a NRPS should aid efforts at swapping NRPS domains between assembly lines.

Dimeric structure of the six-domain VibF subunit of vibriobactin synthetase: mutant domain activity regain and ultracentrifugation studies.

Nonribosomal peptide synthetases (NRPS), fatty acid synthases (FAS), and polyketide sythases (PKS) are multimodular enzymatic assembly lines utilized in natural product biosynthesis. Previous data on FAS and PKS subunits have indicated that they are homodimers and that some of their catalytic functions can work in trans. When NRPS assembly lines have been probed for comparable formation of stable oligomers, no evidence had been forthcoming that species other than monomer forms were active. In this work we focus on the six-domain (Cy1-Cy2-A-C1-PCP-C2) enzyme VibF from the vibriobactin synthetase assembly line, which contains three other proteins, VibB, VibE, and VibH, that--when purified and mixed with VibF and the substrates ATP, threonine, 2,3-dihydroxybenzoate (DHB), and norspermidine--produce the iron chelator vibriobactin. Using a deletion of the Cy1 domain and separate inactivating mutations in the Cy2, A, PCP, and C2 domains of VibF, we report regain of catalytic activity upon mutant protein mixing that argues for heterodimer formation, stable for hundreds to thousands of catalytic cycles, with acyl chain processing and transfer around blocked domains. Ultracentrifugation data likewise confirm a dimeric structure for VibF and establish that domains within NRPS dimeric modules can act on acyl chains in trans. The results described here are the first indication for an NRPS subunit that homodimerization can occur and that there is a continuum of functional oligomerization states between monomers and dimers in nonribosomal peptide synthetases.

Decreasing the ring size of a cyclic nonribosomal peptide antibiotic by in-frame module deletion in the biosynthetic genes.

Many natural products of therapeutical and biotechnological importance are nonribosomally synthesized peptides. Structural hallmarks of this class of compounds are the occurrence of unusual amino acids, mostly cyclic peptide backbones, and numerous further modifications such as acylation, heterocyclic ring formation, and glycosylation. Because of their structural complexity, chemical synthesis is usually an unattractive route to these molecules. In contrast, genetic engineering of the biosynthesis genes emerges as a potentially powerful approach to the combinatorial biosynthesis of useful analogues of the lead compounds. Nonribosomal peptide synthetases (NRPSs) carry out a sequential multistep assembly and modification of the peptides in a thiotemplate process described by the multiple carrier model. The modular architecture of NRPSs suggests straightforward methods for the reprogramming of these enzymes by exchange of catalytic subunits. However, many of the reported engineering attempts faced low product yields or even inactive hybrid enzymes. Using a new approach to obtain hybrid NRPSs, we show here that the deletion of an entire module in an NRPS assembly line caused the secretion of the predicted peptide antibiotic variant with a decreased ring size. Furthermore, a module exchange resulted in a significantly higher product yield than that observed in previous studies.

Genetics and assembly line enzymology of siderophore biosynthesis in bacteria.

The regulatory logic of siderophore biosynthetic genes in bacteria involves the universal repressor Fur, which acts together with iron as a negative regulator. However in other bacteria, in addition to the Fur-mediated mechanism of regulation, there is a concurrent positive regulation of iron transport and siderophore biosynthetic genes that occurs under conditions of iron deprivation. Despite these regulatory differences the mechanisms of siderophore biosynthesis follow the same fundamental enzymatic logic, which involves a series of elongating acyl-S-enzyme intermediates on multimodular protein assembly lines: nonribosomal peptide synthetases (NRPS). A substantial variety of siderophore structures are produced from similar NRPS assembly lines, and variation can come in the choice of the phenolic acid selected as the N-cap, the tailoring of amino acid residues during chain elongation, the mode of chain termination, and the nature of the capturing nucleophile of the siderophore acyl chain being released. Of course the specific parts that get assembled in a given bacterium may reflect a combination of the inventory of biosynthetic and tailoring gene clusters available. This modular assembly logic can account for all known siderophores. The ability to mix and match domains within modules and to swap modules themselves is likely to be an ongoing process in combinatorial biosynthesis. NRPS evolution will try out new combinations of chain initiation, elongation and tailoring, and termination steps, possibly by genetic exchange with other microorganisms and/or within the same bacterium, to create new variants of iron-chelating siderophores that can fit a particular niche for the producer bacterium.

Substrate recognition and selection by the initiation module PheATE of gramicidin S synthetase.

The initiation module of non-ribosomal peptide synthetases (NRPS) selects and activates the first amino acid and serves as the aminoacyl donor in the first peptide bond-forming step of the NRPS assembly line. The gramicidin S synthetase initiation module (PheATE) is a three-domain subunit, recognizing L-phenylalanine (L-Phe) and activating it (by adenylation domain) as tightly bound L-phenylalanyl-adenosine-5'-monophosphate diester (L-Phe-AMP), transferring it to the HS-phosphopantetheine arm of the holo-thiolation (holo-T) domain, and then epimerizing it (by epimerization domain) to the D-Phe-S-4'-Ppant-acyl enzyme. In this study, we have assayed the selectivity of the PheATE adenylation domain with a number of proteinogenic amino acids and observed that three additional amino acids, L-Tyr, L-Trp, and L-Leu, were activated to the aminoacyl-AMPs and transferred to the HS-phosphopantetheine arm of the holo-T domain. Hydrolytic editing of noncognate aminoacyl-AMPs and/or aminoacyl-S-4'-Ppant-acyl enzymes by the enzyme was not observed by three different assays for adenylation domain function. The microscopic reaction rates and thermodynamic equilibrium constants obtained from single-turnover studies of reactions of L-Phe, L-Trp, L-Tyr, and L-Leu with holoPheATE allowed us to construct free energy profiles for the reactions, revealing the kinetic and thermodynamic basis for substrate recognition and selection. In particular, the rates of epimerization of the L-aminoacyl-S-enzyme to the D-aminoacyl-S-enzyme intermediate showed reductions of 245-, 300-, and 540-fold for L-Trp, L-Tyr, and L-Leu respectively, suggesting that the epimerization domain is an important gatekeeper for generation of the D-Phe-S-enzyme that starts gramicidin S chain growth.

In Vitro Reconstitution of the Pseudomonas aeruginosa Nonribosomal Peptide Synthesis of Pyochelin: Characterization of Backbone Tailoring Thiazoline Reductase and N-Methyltransferase Activities

During iron starvation the Gram-negative pathogenic bacterium Pseudomonas aeruginosa makes the nonribosomal peptide siderophore pyochelin by a four protein, 11 domain assembly line, involving a cascade of acyl-S-enzyme intermediates on the PchE and PchF subunits that are elongated, heterocyclized, reduced, and N-methylated before release. Purified PchG is shown to be an NADPH-dependent reductase for the hydroxyphenylbisthiazoline-S-PchF acyl enzyme, regiospecifically converting one of the dihydroheterocyclic thiazoline rings to a thiazolidine. The K(m) for the PchG protein is 1 microM, and the k(cat) for throughput to pyochelin is 2 min(-1). The nitrogen of the newly generated thiazolidine ring can be N-methylated upon addition of SAM, to yield the mature pyochelin chain still tethered as a pyochelinyl-S-PchF at the PCP domain. A presumed methyltransferase (MT) domain embedded in the PchF subunit catalyzes this N-methylation. Mutation of a conserved G to R in the MT core motif abolishes MT activity and subsequent chain release from PchF. The thioesterase (TE) domain of PchF catalyzes hydrolytic release of the fully mature pyochelinyl chain to produce the pyochelin siderophore at a rate of 2 min(-1), at least 30-40-fold faster than in the absence of hydroxyphenylbisthiazolinyl-COOH (HPTT-COOH) chain reduction and N-methylation. A mutation in the PchF TE domain does not catalyze autodeacylation and release of the pyochelinyl-S-enzyme. Thus, full reconstitution of the nonribosomal peptide synthetase assembly line by purified protein components has been obtained for production of this tandem bisheterocyclic siderophore.

Chain Termination Steps in Nonribosomal Peptide Synthetase Assembly Lines: Directed Acyl-S-Enzyme Breakdown in Antibiotic and Siderophore Biosynthesis

Chain Termination Steps in Nonribosomal Peptide Synthetase Assembly Lines: Directed Acyl-S-Enzyme Breakdown in Antibiotic and Siderophore Biosynthesis

Cover Picture

The cover picture shows a false-color image of wild-type Escherichia coli DH5(α) cells growing on an agar plate containing the indicator chromeazurol S. The siderophore enterobactin is secreted by iron-starved colonies, resulting in FeIII-depleted, decolorized halos. The inset scheme shows the pathway for enterobactin biosynthesis from 2,3-dihydroxybenzoic acid (2,3-DHB) and L-serine by the nonribosomal peptide synthetase (NRPS) enzymes EntE, EntB, and EntF. These enzymes catalyze the formation of peptide bonds without mRNA templates and have a modular structure. A functional module consists of an adenylation (A), a thiolation (T), and a condensation (C) domain. The terminal thioesterase (TE) domain of EntF catalyzes elongation and cyclotrimerization to the final product. Further details about the diversity and function of the chain-releasing C-terminal domains of NRPS assembly lines can be found in the Minireview by C. T. Walsh et al. on p. 99 ff.

Cover Picture

The cover picture shows a false-color image of wild-type Escherichia coli DH5(α) cells growing on an agar plate containing the indicator chromeazurol S. The siderophore enterobactin is secreted by iron-starved colonies, resulting in FeIII-depleted, decolorized halos. The inset scheme shows the pathway for enterobactin biosynthesis from 2,3-dihydroxybenzoic acid (2,3-DHB) and L-serine by the nonribosomal peptide synthetase (NRPS) enzymes EntE, EntB, and EntF. These enzymes catalyze the formation of peptide bonds without mRNA templates and have a modular structure. A functional module consists of an adenylation (A), a thiolation (T), and a condensation (C) domain. The terminal thioesterase (TE) domain of EntF catalyzes elongation and cyclotrimerization to the final product. Further details about the diversity and function of the chain-releasing C-terminal domains of NRPS assembly lines can be found in the Minireview by C. T. Walsh et al. on p. 99 ff.

Chain Termination Steps in Nonribosomal Peptide Synthetase Assembly Lines: Directed Acyl-S-Enzyme Breakdown in Antibiotic and Siderophore Biosynthesis

The multidomain enzymes that biosynthesize important natural peptide products such as cyclosporin and vancomycin carry the growing biopolymer chains covalently attached as thioesters. For the mature chain to be released from the enzyme, this thioester must be cleaved, a task that falls to specialized enzymatic domains that can hydrolyze these thioesters, reduce them, or macrocyclize the substrate to form a new amide bond (schematically shown for the tyrocidine thioesterase). This Minireview examines these terminating domains and their mechanisms. NAC=N-acetylcysteaminyl.

The EntF and EntE adenylation domains of Escherichia coli enterobactin synthetase: sequestration and selectivity in acyl-AMP transfers to thiolation domain cosubstrates.

Enterobactin, the tris-(N-(2,3-dihydroxybenzoyl)serine) trilactone siderophore of Escherichia coli, is synthesized by a three-protein (EntE, B, F) six-module nonribosomal peptide synthetase (NRPS). In this work, the 142-kDa four-domain protein EntF was bisected into two double-domain fragments: a 108-kDa condensation and adenylation construct, EntF C-A, and a 37-kDa peptidyl carrier protein (PCP) and thioesterase protein, EntF PCP-TE. The adenylation domain activity of EntF C-A formed seryl-AMP but lost the ability to transfer the seryl moiety to the cognate EntF PCP-TE in trans. Seryl transfer to heterologous PCP protein fragments, the SrfB1 PCP from surfactin synthetase and Ybt PCP1 from yersiniabactin synthetase, was observed at rates of 0.5 min(-1) and 0.01 min(-1), respectively. The possibility that these slow acylation rates reflected dissociation of acyl/aminoacyl-AMP followed by adventitious thiolation by the heterologous PCPs in solution was addressed by measuring catalytic turnover of pyrophosphate (PP(i)) released from the adenylation domain. The holo SrfB1 PCP protein as well as Ybt PCP1 did not stimulate an increase in PP(i) release from EntF C-A or EntE. In this light, aminoacylations in trans between A and PCP domain fragments of NRPS assembly lines must be subjected to kinetic scrutiny to determine whether transfer is truly between protein domains or results from slow aminoacyl-AMP release and subsequent nonenzymatic thiol capture.