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.     

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.     

Synthesis of (di)adenosine polyphosphates by non-ribosomal peptide synthetases (NRPS)

In response to nutritional stress conditions, Bacillus brevis produces the cyclodecapeptide antibiotic tyrocidine via tyrocidine synthetase, a multifunctional non-ribosomal peptide synthetase. The apo-form of tyrocidine synthetase 1 forms adenosine (5')tetraphospho(5')adenosine, when incubated with MgATP(2-), amino acid and inorganic pyrophosphatase. The synthesis is an intrinsic property of the adenylation domain, is strictly dependent upon the amino acid, and proceeds from a reverse reaction of adenylate formation involving a second ATP molecule. In the presence of tri- or tetrapolyphosphate preferential synthesis of adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate occurs, respectively. A potential involvement of adenosine (5')-n-phospho(5')adenosine in the regulation of the biosynthetic process has been suggested.     

Genetic evidence for a role of thioesterase domains, integrated in or associated with peptide synthetases, in non-ribosomal peptide biosynthesis in Bacillus subtilis

Next to almost all prokaryotic operons encoding peptide synthetases, which are involved in the nonribosomal synthesis of peptide antibiotics, distinct genes have been detected that encode proteins with strong sequence similarity to type II fatty acid thioesterases of vertebrate origin. Furthermore, sequence analysis of bacterial and fungal peptide synthetases has revealed a region at the C-terminal end of modules that are responsible for adding the last amino acid to the peptide antibiotics; that region also exhibits significant similarities to thioesterases. In order to investigate the function of these putative thioesterases in non-ribosomal peptide synthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis, srfA fragments encoding the thioesterase domain of the surfactin synthetase 3 and the thioesterase-like protein SrfA-TE were deleted. This led to a 97 and 84% reduction of the in vivo surfactin production, respectively. In the double mutant, however, no surfaction production was detectable. These findings demonstrate for the first time that the C-terminal thioesterase domains and the SrfA-TE protein are directly involved in nonribosomal peptide biosynthesis. Received: 30 September 1997 / Accepted: 4 December 1997     

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 revealing an unusual N-terminal region in a stand-alone non-ribosomal peptide synthetase involved in the biosynthesis of ramoplanins

Ramoplanins produced by Actinoplanes are new structural class of lipopeptide and are currently in phase III clinical trials for the prevention of vancomycin-resistant enterococcal infections. The depsipeptide structures of ramoplanins are synthesized by non-ribosomal peptide synthetases (NRPS). Romo-orf17, a stand-alone NRPS, is responsible for the recruitment of Thr into the linear NRPS pathways for which the corresponding adenylation domain is absent. Here, systematical gene inactivation and complementation have been carried out in a Actinoplanes sp. using homologous recombination and site-specific integration methods. A hybrid gene coding for the N-terminal region of the stand-alone NRPS and the A-PCP domains of a heterologous NRPS restored production of ramoplanins. The results elucidate the unusual N-terminal region which is essential for the biosynthesis of ramoplanins.     

Deep sequencing of non-ribosomal peptide synthetases and polyketide synthases from the microbiomes of Australian marine sponges

The biosynthesis of non-ribosomal peptide and polyketide natural products is facilitated by multimodular enzymes that contain domains responsible for the sequential condensation of amino and carboxylic subunits. These conserved domains provide molecular targets for the discovery of natural products from microbial metagenomes. This study demonstrates the application of tag-encoded FLX amplicon pyrosequencing (TEFAP) targeting non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes as a method for determining the identity and diversity of natural product biosynthesis genes. To validate this approach, we assessed the diversity of NRPS and PKS genes within the microbiomes of six Australian marine sponge species using both TEFAP and metagenomic whole-genome shotgun sequencing approaches. The TEFAP approach identified 100 novel ketosynthase (KS) domain sequences and 400 novel condensation domain sequences within the microbiomes of the six sponges. The diversity of KS domains within the microbiome of a single sponge species Scopalina sp. exceeded that of any previously surveyed marine sponge. Furthermore, this study represented the first to target the condensation domain from NRPS biosynthesis and resulted in the identification of a novel condensation domain lineage. This study highlights the untapped potential of Australian marine sponges for the isolation of novel bioactive natural products. Furthermore, this study demonstrates that TEFAP approaches can be applied to functional genes, involved in natural product biosynthesis, as a tool to aid natural product discovery. It is envisaged that this approach will be used across multiple environments, offering an insight into the biological processes that influence the production of secondary metabolites.     

Bioactive compounds synthesized by non-ribosomal peptide synthetases and type-I polyketide synthases discovered through genome-mining and metagenomics

Non-ribosomal peptide synthetases (NRPS) and type-I polyketide synthases (PKS-I) are multimodular enzymes involved in biosynthesis of oligopeptide and polyketide secondary metabolites produced by microorganisms such as bacteria and fungi. New findings regarding the mechanisms underlying NRPS and PKS-I evolution illustrate how microorganisms expand their metabolic potential. During the last decade rapid development of bioinformatics tools as well as improved sequencing and annotation of microbial genomes led to discovery of novel bioactive compounds synthesized by NRPS and PKS-I through genome-mining. Taking advantage of these technological developments metagenomics is a fast growing research field which directly studies microbial genomes or specific gene groups and their products. Discovery of novel bioactive compounds synthesized by NRPS and PKS-I will certainly be accelerated through metagenomics, allowing the exploitation of so far untapped microbial resources in biotechnology and medicine.     

PCR detection of novel non-ribosomal peptide synthetase genes in lipopeptide-producing Pseudomonas

Bacterial lipopeptides (LPs) are a diverse group of secondary metabolites synthesized through one or more non-ribosomal peptide synthetases (NRPSs). In certain genera, such as Pseudomonas and Bacillus, these enzyme systems are often involved in synthesizing biosurfactants or antimicrobial compounds. Several different types of LPs have been reported for non-pathogenic plant-associated Pseudomonas. Focusing on this group of bacteria, we devised and validated a PCR method to detect novel LP-synthesizing NRPS genes by targeting their lipoinitiation and tandem thioesterase domains, thus avoiding amplification of genes for non-LP metabolites, such as the pyoverdine siderophores present in all fluorescent Pseudomonas. This approach enabled detection of as yet unknown NRPS genes in strains producing viscosin, viscosinamide, WLIP, or lokisin. Furthermore, it proved valuable to identify novel candidate LP producers among Pseudomonas rhizosphere isolates. By phylogenetic analysis of these amplicons, several of the corresponding NRPS genes can be tentatively assigned to the viscosin, amphisin, or entolysin biosynthetic groups, while some others may represent novel NRPS systems.     

Ironing out siderophore biosynthesis: a review of non-ribosomal peptide synthetase (NRPS)-independent siderophore synthetases

Iron is required for microbial growth and proliferation. To survive in low-iron environments, some microorganisms secrete ferric iron chelators called siderophores. Siderophore biosynthesis occurs via two pathways: the non-ribosomal peptide synthetase (NRPS) pathway and the NRPS-independent siderophore (NIS) synthetase pathway. NIS enzymes function by adenylating a carboxylic acid substrate, typically citrate, or a derivative, followed by nucleophilic capture of an amine or alcohol and displacement of a citryl intermediate. In this review, we summarize recent advances in NIS biochemistry with a particular focus on structural biology and confirm the classification of NIS enzymes into Types A, A’, B, and C based on substrate specificity. Based on a phylogenetic analysis, we also propose a new subclass of NIS enzymes, Type C’, responsible for dimerization and macrocyclization of complex and substituted amine or amide intermediates. Finally, we describe the role of NIS enzymes in virulence of pathogenic microbes and discuss NIS inhibitors as potential anti-microbial agents.     

Understanding the crucial interactions between Cytochrome P450s and non-ribosomal peptide synthetases during glycopeptide antibiotic biosynthesis

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The expression of selected non-ribosomal peptide synthetases in Aspergillus fumigatus is controlled by the availability of free iron

Three non-ribosomal peptide synthetase genes, termed sidD, sidC and sidE, have been identified in Aspergillus fumigatus. Gene expression analysis by RT-PCR confirms that expression of both sidD and C was reduced by up to 90% under iron-replete conditions indicative of a likely role in siderophore biosynthesis. SidE expression was less sensitive to iron levels. In addition, two proteins purified from mycelia grown under iron-limiting conditions corresponded to SidD ( approximately 200 kDa) and SidC (496 kDa) as determined by MALDI ToF peptide mass fingerprinting and MALDI LIFT-ToF/ToF. Siderophore synthetases are unique in bacteria and fungi and represent an attractive target for antimicrobial chemotherapy.     

Structural aspects of non-ribosomal peptide biosynthesis

Small peptides have powerful biological activities ranging from antibiotic to immune suppression. These peptides are synthesized by non-ribosomal peptide synthetases (NRPS). Structural understanding of NRPS took a huge leap forward in 2002; this information has led to several detailed biochemical studies and further structural studies. NRPS are complex molecular machines composed of multiple modules and each module contains several autonomously folded catalytic domains. Structural studies have largely focused on individual domains, isolated from the context of the multienzyme. Biochemical studies have looked at individual domains, isolated whole modules and intact NRPS, and the combined data begin to allow us to visualize the process of peptide assembly by NRPS.     

Isoprostanes: novel bioactive products of lipid peroxidation

Isoprostanes, are a novel group of prostaglandin-like compounds that are biosynthesised from esterified polyunsaturated fatty acid (PUFA) through a non-enzymatic free radical-catalysed reaction. Several of these compounds possess potent biological activity, as evidenced mainly through their pulmonary and renal vasoconstrictive effects, and have short half-lives. It has been shown that isoprostanes act as full or partial agonists through thromboxane receptors. Both human and experimental studies have indicated associations of isoprostanes and severe inflammatory conditions, ischemia-reperfusion, diabetes and atherosclerosis. Reports have shown that F₂-isoprostanes are authentic biomarkers of lipid peroxidation and can be used as potential in vivo indicators of oxidant stress in various clinical conditions, as well as in evaluations of antioxidants or drugs for their free radical-scavenging properties.

Higher levels of F₂-isoprostanes have been found in the normal human pregnancy compared to non-pregnancy, but their physiological role has not been well studied so far. Since bioactive F₂-isoprostanes are continuously formed in various tissues and large amounts of these potent compounds are found unmetabolised in their free acid form in the urine in normal basal conditions with a wide inter-individual variation, their role in the regulation of normal physiological functions could be of further biological interest, but has yet to be disclosed. Their potent biological activity has attracted great attention among scientists, since these compounds are found in humans and animals in both physiological and pathological conditions and can be used as reliable biomarkers of lipid peroxidation.     

Engineering cyanobacteria to generate high-value products

Although many microorganisms have been used for the bioindustrial generation of valuable metabolites, the productive potential of cyanobacterial species has remained largely unexplored. Cyanobacteria possess several advantages as organisms for bioindustrial processes, including simple input requirements, tolerance of marginal agricultural environments, rapid genetics, and carbon-neutral applications that could be leveraged to address global climate change concerns. Here, we review recent research involving the engineering of cyanobacterial species for the production of valuable bioindustrial compounds, including natural cyanobacterial products (e.g. sugars and isoprene), biofuels (e.g. alcohols, alkanes and hydrogen), and other commodity chemicals. Biological and economic obstacles to scaled cyanobacterial production are highlighted, and methods for increasing cyanobacterial production efficiencies are discussed.     

A novel reporter system for bacterial and mammalian cells based on the non-ribosomal peptide indigoidine

The biosynthesis of non-ribosomal peptides, many of which have pharmaceutical activities, is an evolutionary privilege of microorganisms. Capitalizing on the universal set of the Streptomyces lavendulae non-ribosomal peptide synthase BpsA and the Streptomyces verticillus 4'-phosphopantetheinyl transferase Svp, we have engineered Escherichia coli as well as mammalian cells, including human stem cells, to produce the blue 3,3'-bipyridyl pigment keto-indigoidine and the reduced colorless but fluorescent leuco-isoform. Detailed characterization of a tailored substrate-free chromogenic assay and FACS analysis showed that indigoidine's blue color and fluorescence could be reliably profiled in bacteria and mammalian cells using standard multiwell-compatible detection equipment. Besides serving as an inexpensive, reliable, versatile and easy-to-assay cross-kingdom reporter system, the potential of having mammalian cells produce non-ribosomal peptides, preferably ones with biopharmaceutical activities, may provide novel treatment opportunities in future gene- and cell-based therapies.     

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.     

A new phage-display tumor-homing peptide fused to antiangiogenic peptide generates a novel bioactive molecule with antimelanoma activity

Phage-display peptide libraries have been widely used to identify specific peptides targeting in vivo tumor cells and the tumor vasculature and playing an important role in the discovery of antitumor bioactive peptides. In the present work, we identified a new melanoma-homing peptide, (-CVNHPAFAC-), using a C7C phage-display library directed to the developing tumor in syngeneic mice. Phage were able to preferentially target melanoma in vivo, with an affinity about 50-fold greater than that with normal tissue, and the respective synthesized peptide displaced the corresponding phage from the tumor. A preferential binding to endothelial cells rather than to melanoma cells was seen in cell ELISA, suggesting that the peptide is directed to the melanoma vasculature. Furthermore, the peptide was able to bind to human sonic hedgehog, a protein involved in the development of many types of human cancers. Using a new peptide approach therapy, we coupled the cyclic peptide to another peptide, HTMYYHHYQHHL-NH(2), a known antagonist of VEGFR-2 receptor, using the GYG linker. The full peptide CVNHPAFACGYGHTMYYHHYQHHL-NH(2) was effective in delaying tumor growth (P < 0.05) and increasing animal survival when injected systemically, whereas a scramble-homing peptide containing the same antagonist did not have any effect. This is the first report on the synthesis of a tumor-homing peptide coupled to antiangiogenic peptide as a new anticancer therapeutics.     

Biosynthetic engineering of nonribosomal peptide synthetases

From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium‐dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.