Evolution-inspired engineering of nonribosomal peptide synthetases.

Many clinically used drugs are derived from or inspired by bacterial natural products that often are produced through nonribosomal peptide synthetases (NRPSs), megasynthetases that activate and join individual amino acids in an assembly line fashion. In this work, we describe a detailed phylogenetic analysis of several bacterial NRPSs that led to the identification of yet undescribed recombination sites within the thiolation (T) domain that can be used for NRPS engineering. We then developed an evolution-inspired "eXchange Unit between T domains" (XUT) approach, which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

Modular catalytic activity of nonribosomal peptide synthetases depends on the dynamic interaction between adenylation and condensation domains.

Nonribosomal peptide synthetases (NRPSs) are large multidomain enzymes for the synthesis of a variety of bioactive peptides in a modular and pipelined fashion. Here, we investigated how the condensation (C) domain and the adenylation (A) domain cooperate with each other for the efficient catalytic activity in microcystin NRPS modules. We solved two crystal structures of the microcystin NRPS modules, representing two different conformations in the NRPS catalytic cycle. Our data reveal that the dynamic interaction between the C and the A domains in these modules is mediated by the conserved "RXGR" motif, and this interaction is important for the adenylation activity. Furthermore, the "RXGR" motif-mediated dynamic interaction and its functional regulation are prevalent in different NRPSs modules possessing both the A and the C domains. This study provides new insights into the catalytic mechanism of NRPSs and their engineering strategy for synthetic peptides with different structures and properties.

Subdomain dynamics enable chemical chain reactions in non-ribosomal peptide synthetases.

Many peptide-derived natural products are produced by non-ribosomal peptide synthetases (NRPSs) in an assembly-line fashion. Each amino acid is coupled to a designated peptidyl carrier protein (PCP) through two distinct reactions catalysed sequentially by the single active site of the adenylation domain (A-domain). Accumulating evidence suggests that large-amplitude structural changes occur in different NRPS states; yet how these molecular machines orchestrate such biochemical sequences has remained elusive. Here, using single-molecule Förster resonance energy transfer, we show that the A-domain of gramicidin S synthetase I adopts structurally extended and functionally obligatory conformations for alternating between adenylation and thioester-formation structures during enzymatic cycles. Complementary biochemical, computational and small-angle X-ray scattering studies reveal interconversion among these three conformations as intrinsic and hierarchical where intra-A-domain organizations propagate to remodel inter-A-PCP didomain configurations during catalysis. The tight kinetic coupling between structural transitions and enzymatic transformations is quantified, and how the gramicidin S synthetase I A-domain utilizes its inherent conformational dynamics to drive directional biosynthesis with a flexibly linked PCP domain is revealed.

A dissected non-ribosomal peptide synthetase maintains activity.

Non-ribosomal peptide synthetases (NRPSs) generate chemically complex compounds and their modular architecture suggests that changing their domain organization can predictably alter their products. Ebony, a small three-domain NRPS, catalyzes the formation of ß-alanine containing amides from biogenic amines. To examine the necessity of interdomain interactions, we modeled and docked domains of Ebony to reveal potential interfaces between them. Testing the same domain combinations in vitro showed that 8 % of activity was preserved after Ebony was dissected into a di-domain and a detached C-terminal domain, suggesting that sufficient interaction was maintained after dissection. Our work creates a model to identify domain interfaces necessary for catalysis, an important step toward utilizing Ebony as a combinatorial engineering platform for novel amides.

Peptide mixed phosphonates for covalent complex formation with thioesterases in nonribosomal peptide synthetases.

Natural macrocyclic peptides derived from microorganisms are medicinal resources that are important for the development of new therapeutic agents. Most of these molecules are biosynthesized by a nonribosomal peptide synthetase (NRPS). The thioesterase (TE) domain in NRPS is responsible for the macrocyclization of mature linear peptide thioesters in a final biosynthetic step. NRPS-TEs can cyclize synthetic linear peptide analogs and can be utilized as biocatalysts for the preparation of natural product derivatives. Although the structures and enzymatic activities of TEs have been investigated, the substrate recognition and substrate-TE interaction during the macrocyclization step are still unknown. To understand the TE-mediated macrocyclization, here we report the development of a substrate-based analog with mixed phosphonate warheads, which can react irreversibly with the Ser residue at the active site of TE. We have demonstrated that the tyrocidine A linear peptide (TLP) with a p-nitrophenyl phosphonate (PNP) enables efficient complex formation with tyrocidine synthetase C (TycC)-TE containing tyrocidine synthetase.

Structure-guided design and synthesis of ATP-competitive N-acyl-substituted sulfamide d-alanine-d-alanine ligase inhibitors.

d-Alanine-d-alanine ligase (Ddl) catalyses the ATP-dependent formation of d-Ala-d-Ala, a critical component in bacterial cell wall biosynthesis and is a validated target for new antimicrobial agents. Here, we describe the structure-guided design, synthesis, and evaluation of ATP-competitive N-acyl-substituted sulfamides 27-36, 42, 46, 47 as inhibitors of Staphylococcus aureus Ddl (SaDdl). A crystal structure of SaDdl complexed with ATP and d-Ala-d-Ala (PDB: 7U9K) identified ATP-mimetic 8 as an initial scaffold for further inhibitor design. Evaluation of 8 in SaDdl enzyme inhibition assays revealed the ability to reduce enzyme activity to 72 ± 8 % (IC50 = 1.6 mM). The sulfamide linker of 8 was extended with 2-(4-methoxyphenyl)ethanol to give 29, to investigate further interactions with the d-Ala pocket of SaDdl, as predicted by molecular docking. This compound reduced enzyme activity to 89 ± 1 %, with replacement of the 4-methoxyphenyl group in 29 with alternative phenyl substituents (27, 28, 31-33, 35, 36) failing to significantly improve on this (80-89 % remaining enzyme activity). Exchanging these phenyl substituents with selected heterocycles (42, 46, 47) did improve activity, with the most active compound (42) reducing SaDdl activity to 70 ± 1 % (IC50 = 1.7 mM), which compares favourably to the FDA-approved inhibitor d-cycloserine (DCS) (IC50 = 0.1 mM). To the best of our knowledge, this is the first reported study of bisubstrate SaDdl inhibitors.

Mur ligase F as a new target for the flavonoids quercitrin, myricetin, and (-)-epicatechin.

MurC, D, E, and F are ATP-dependent ligases involved in the stepwise assembly of the tetrapeptide stem of forming peptidoglycan. As highly conserved targets found exclusively in bacterial cells, they are of significant interest for antibacterial drug discovery. In this study, we employed a computer-aided molecular design approach to identify potential inhibitors of MurF. A biochemical inhibition assay was conducted, screening twenty-four flavonoids and related compounds against MurC-F, resulting in the identification of quercitrin, myricetin, and (-)-epicatechin as MurF inhibitors with IC50 values of 143 µM, 139 µM, and 92 µM, respectively. Notably, (-)-epicatechin demonstrated mixed type inhibition with ATP and uncompetitive inhibition with D-Ala-D-Ala dipeptide and UM3DAP substrates. Furthermore, in silico analysis using Sitemap and subsequent docking analysis using Glide revealed two plausible binding sites for (-)-epicatechin. The study also investigated the crucial structural features required for activity, with a particular focus on the substitution pattern and hydroxyl group positions, which were found to be important for the activity. The study highlights the significance of computational approaches in targeting essential enzymes involved in bacterial peptidoglycan synthesis.

Comparative functional and structural analysis of Pseudomonas aeruginosa d-alanine-d-alanine ligase isoforms as prospective antibiotic targets.

Pseudomonas aeruginosa is a major human pathogen in the healthcare setting. The emergence of multi-drug-resistant and extensive drug-resistant P. aeruginosa is of great concern, and clearly indicates that new alternatives to current first-line antibiotics are required in the future. Inhibition of d-alanine-d-alanine production presents as a promising avenue as it is a key component in the essential process of cell wall biosynthesis. In P. aeruginosa, d-alanine-d-alanine production is facilitated by two isoforms, d-alanine-d-alanine ligase A (PaDdlA) and d-alanine-d-alanine ligase B (PaDdlA), but neither enzyme has been individually characterised to date. Here, we present the functional and structural characterisation of PaDdlA and PaDdlB, and assess their potential as antibiotic targets. This was achieved using a combination of in vitro enzyme-activity assays and X-ray crystallography. The former revealed that both isoforms effectively catalyse d-alanine-d-alanine production with near identical efficiency, and that this is effectively disrupted by the model d-alanine-d-alanine ligase inhibitor, d-cycloserine. Next, each isoform was co-crystallised with ATP and either d-alanine-d-alanine or d-cycloserine, allowing direct comparison of the key structural features. Both isoforms possess the same structural architecture and share a high level of conservation within the active site. Although residues forming the d-alanine pocket are completely conserved, the ATP-binding pocket possesses several amino acid substitutions resulting in a differing chemical environment around the ATP adenine base. Together, these findings support that the discovery of dual PaDdlA/PaDdlB competitive inhibitors is a viable approach for developing new antibiotics against P. aeruginosa.

Guidelines for Optimizing Type S Nonribosomal Peptide Synthetases.

Bacterial biosynthetic assembly lines, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), play a crucial role in the synthesis of natural products that have significant therapeutic potential. The ability to engineer these biosynthetic assembly lines offers opportunities to produce artificial nonribosomal peptides, polyketides, and their hybrids with improved properties. In this study, we introduced a synthetic NRPS variant, termed type S NRPS, which simplifies the engineering process and enables biocombinatorial approaches for generating nonribosomal peptide libraries in a parallelized high-throughput manner. However, initial generations of type S NRPSs exhibited a bottleneck that led to significantly reduced production yields. To address this challenge, we employed two optimization strategies. First, we truncated SYNZIPs from the N- and/or C-terminus of the NRPS. SYNZIPs comprise a large set of well-characterized synthetic protein interaction reagents. Second, we incorporated a structurally flexible glycine-serine linker between the NRPS protein and the attached SYNZIP, aiming to improve dynamic domain-domain interactions. Through an iterative optimization process, we achieved remarkable improvements in production yields, with titer increases of up to 55-fold compared to the nonoptimized counterparts. These optimizations successfully restored production levels of type S NRPSs to those observed in wild-type NRPSs and even surpassed them. Overall, our findings demonstrate the potential of engineering bacterial biosynthetic assembly lines for the production of artificial nonribosomal peptides. In addition, optimizing the SYNZIP toolbox can have valuable implications for diverse applications in synthetic biology, such as metabolic engineering, cell signaling studies, or engineering of other multienzyme complexes, such as PKSs.

Biomimetic engineering of nonribosomal peptide synthesis.

Nonribosomal peptides (NRPs) have gained attention due to their diverse biological activities and potential applications in medicine and agriculture. The natural diversity of NRPs is a result of evolutionary processes that have occurred over millions of years. Recent studies have shed light on the mechanisms by which nonribosomal peptide synthetases (NRPSs) evolve, including gene duplication, recombination, and horizontal transfer. Mimicking natural evolution could be a useful strategy for engineering NRPSs to produce novel compounds with desired properties. Furthermore, the emergence of antibiotic-resistant bacteria has highlighted the urgent need for new drugs, and NRPs represent a promising avenue for drug discovery. This review discusses the engineering potential of NRPSs in light of their evolutionary history.

Biosynthetic diversification of non-ribosomal peptides through activity-based protein profiling of adenylation domains.

We describe activity-based protein profiling for analyzing the adenylation domains of non-ribosomal peptide synthetases (ABPP-NRPS) in bacterial proteomes. Using a range of non-proteoinogenic amino acid sulfamoyladenosines, the competitive format of ABPP-NRPS provided substrate tolerance toward non-proteinogenic amino acids. When coupled with precursor-directed biosynthesis, a non-proteinogenic amino acid (O-allyl-L-serine) was successfully incorporated into gramicidin S.

Not always an innocent bystander: the impact of stabilised phosphopantetheine moieties when studying nonribosomal peptide biosynthesis.

Nonribosomal peptide synthetases produce many important peptide natural products and are centred around carrier proteins (CPs) that deliver intermediates to various catalytic domains. We show that the replacement of CP substrate thioesters by stabilised ester analogues leads to active condensation domain complexes, whereas amide stabilisation generates non-functional complexes.

Engineered Biosynthesis through the Adenylation Domains from Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases, consisted of multiple catalytic domains, are involved in the biosynthesis of an important family of bioactive natural products in a coordinated manner. Among the functional domains, adenylation domains are specifically responsible for recognizing carboxylic acid building blocks and synthesizing aminoacyl adenylates. Given their critical roles in the biosynthesis of the growing peptide, A-domains are also referred to as the "gatekeeper". In this review, very recent developments on the A-domains from NRPSs are reviewed to expand the fundamental knowledge of the A domain, including knowledge on the structures, functions, and molecular interactions. Several recent examples were also discussed to highlight the great potential of A-domain engineering. This study should provide a framework for the combinatorial biosynthesis or synthetic biology-driven microbial production of novel nonribosomal peptides.

The Assembly-Line Enzymology of Nonribosomal Peptide Biosynthesis.

Peptide natural products constitute a major class of secondary metabolites produced by microorganisms (mostly bacteria and fungi). In the past several decades, researchers have gained extensive knowledge about nonribosomal peptides (NRPs) generated by ribosome-independent systems, namely, NRP synthetases (NRPSs). NRPSs are multifunctional enzymes consisting of semiautonomous domains that form a peptide backbone. Using a thiotemplate mechanism that employs assembly-line logic with multiple modules, NRPSs activate, tether, and modify amino acid building blocks, sequentially elongating the peptide chain before releasing the complete peptide. Adenylation, thiolation, condensation, and thioesterase domains play central roles in these reactions. This chapter focuses on the current understanding of these central domains in NRPS assembly-line enzymology.

Structural Studies of Modular Nonribosomal Peptide Synthetases.

The non-ribosomal peptide synthetases (NRPSs) are a family of modular enzymes involved in the production of peptide natural products. Not restricted by the constraints of ribosomal peptide and protein production, the NRPSs are able to incorporate unusual amino acids and other suitable building blocks into the final product. The NRPSs operate with an assembly line strategy in which peptide intermediates are covalently tethered to a peptidyl carrier protein and transported to different catalytic domains for the multiple steps in the biosynthesis. Often the carrier and catalytic domains are joined into a single large multidomain protein. This chapter serves to introduce the NRPS enzymes, using the nocardicin NRPS system as an example that highlights many common features to NRPS biochemistry. We then describe recent advances in the structural biology of NRPSs focusing on large multidomain structures that have been determined.

Chemoproteomic Profiling of Adenylation Domain Functions in Gramicidin S-Producing Non-ribosomal Peptide Synthetases.

Many amino acid-containing natural products are biosynthesized by large, multifunctional enzymes known as non-ribosomal peptide synthetases (NRPSs). Adenylation (A) domains in NRPSs are responsible for the incorporation of amino acid building blocks and can be considered as engineering domains; therefore, advanced techniques are required to not only rapidly verify expression and folding, but also accelerate the functional prediction of the A-domains in lysates from native and heterologous systems. We recently developed activity-based protein profiling (ABPP) of NRPSs that offers a simple and robust analytical platform for A-domains and provides insights into their enzyme-substrate specificity. In this chapter, we describe the design and synthesis of these ABPP probes and provide a summary of our work on the development of a series of protocols for labeling, visualizing, and analyzing endogenous NRPSs in complex biological systems.

In Vitro Biochemical Characterization of Excised Macrocyclizing Thioesterase Domains from Non-ribosomal Peptide Synthetases.

Characterization of thioesterases (TEs) is an important step in understanding natural product biosynthesis. Studying non-ribosomal peptide synthetase (NRPS) TEs presents a unique set of challenges with specific cloning and expression issues as well as the challenging synthesis of the thioester peptides substrate required for characterization of the TE. In this method, we describe the cloning and expression of NRPS TEs, the synthesis of thioester peptides, and the in vitro biochemical characterization of the enzyme.

Unraveling Structural Information of Multi-Domain Nonribosomal Peptide Synthetases by Using Photo-Cross-Linking Analysis with Genetic Code Expansion.

Nonribosomal peptide synthetases (NRPSs) are large, multifunctional enzymes that facilitate the stepwise synthesis of modified peptides, many of which serve as important pharmaceutical products. Typically, NRPSs contain one module for the incorporation of one amino acid into the growing peptide chain. A module consists of the domains required for activation, covalent binding, condensation, termination, and optionally modification of the aminoacyl or peptidyl moiety. We here describe a protocol using genetically encoded photo-cross-linking amino acids to probe the 3D architecture of NRPSs by determining spatial proximity constraints. p-benzoyl-L-phenylalanine (BpF) is incorporated at positions of presumed contact interfaces between domains. The covalent cross-link products are visualized by SDS-PAGE-based methods and precisely mapped by tandem mass spectrometry. Originally intended to study the communication (COM) domains, a special pair of docking domains of unknown structure between two interacting subunits of one NRPS system, this cross-linking approach was also found to be useful to interrogate the spatial proximity of domains that are not connected on the level of the primary structure. The presented photo-cross-linking technique thus provides structural insights complementary to those obtained by protein crystallography and reports on the protein in solution.

A Chemoenzymatic Approach to Investigate Cytochrome P450 Cross-Linking in Glycopeptide Antibiotic Biosynthesis.

Glycopeptide antibiotics (GPAs) are important and medically relevant peptide natural products. In the context of antimicrobial resistance (AMR), understanding and manipulating GPA biosynthesis is essential to discover new bioactive derivatives of these peptides. Among all the enzymatic steps in GPA biosynthesis, the most complex occurs during the maturation (cross-linking) of the peptide aglycone. This is achieved-while the peptide remains attached to the nonribosomal peptide synthetase (NRPS) machinery-through the action of a cytochrome P450 (CYP450 or Oxy)-mediated cyclization cascade. There is great interest in understanding the formation of the cross-links between the aromatic side chains in GPAs as this process leads to the cup-shaped aglycone, which is itself a requirement for antibiotic activity. In this regard, the use of in vitro experiments is crucial to study this process. To address the process of peptide cyclization during GPA biosynthesis, a series of peptide substrates and different Oxy enzymes are required. In this chapter, we describe a practical and efficient route for the synthesis of peptidyl-CoAs, the expression of proteins/enzymes involved in the in vitro cyclization assay, the loading of the PCP with peptidyl-CoAs, an optimized CYP450-mediated cyclization cascade and assay workup followed by mass spectrometry (MS) characterization. This in vitro assay affords high conversion to cyclic peptides and demonstrates the tolerance of the P450s for novel GPA precursor peptide substrates.

Cross-Linking of the Nonribosomal Peptide Synthetase Adenylation Domain with a Carrier Protein Using a Pantetheine-Type Probe.

Adenylation domains (A-domains) are responsible for the selective incorporation of carboxylic acid substrates in the biosynthesis of nonribosomal peptides and related natural products. The A-domain transfers an acyl substrate onto its cognate carrier protein (CP). The proper interactions between an A-domain and the cognate CP are important for functional substrate transfer. To stabilize the transient interactions sufficiently for structural analysis of A-domain-CP complex, vinylsulfonamide adenosine inhibitors have been traditionally used as molecular probes. Recently, we have developed an alternative strategy using a synthetic pantetheine-type probe that enables site-specific cross-linking between an A-domain and a CP. In this chapter, we describe the laboratory protocols for this cross-linking reaction.