Several peptides in clinical use are derived from non-ribosomal peptide synthetases (NRPS). In these systems multiple NRPS subunits interact with each other in a specific linear order mediated by ...specific docking domains (DDs), whose structures are not known yet, to synthesize well-defined peptide products. In contrast to classical NRPSs, single-module NRPS subunits responsible for the generation of rhabdopeptide/xenortide-like peptides (RXPs) can act in different order depending on subunit stoichiometry thereby producing peptide libraries. To define the basis for their unusual interaction patterns, we determine the structures of all N-terminal DDs (
DDs) as well as of an
DD-
DD complex and characterize all putative DD interactions thermodynamically for such a system. Key amino acid residues for DD interactions are identified that upon their exchange change the DD affinity and result in predictable changes in peptide production. Recognition rules for DD interactions are identified that also operate in other megasynthase complexes.
Structure elucidation of natural products including the absolute configuration is a complex task that involves different analytical methods like mass spectrometry, NMR spectroscopy, and chemical ...derivation, which are usually performed after the isolation of the compound of interest. Here, a combination of stable isotope labeling of Photorhabdus and Xenorhabdus strains and their transaminase mutants followed by detailed MS analysis enabled the structure elucidation of novel cyclopeptides named GameXPeptides including their absolute configuration in crude extracts without their actual isolation.
Game over—structure solved: A combination of labeling experiments with mass spectrometry results in the reliable determination of the sum formula, the nature of the building blocks, and (for peptide natural products) also the determination of the absolute configuration as exemplified for the novel natural products GameXPeptide A–D (see scheme).
An E. coli strain with deletions in five transaminases (ΔaspC ΔilvE ΔtyrB ΔavtA ΔybfQ) was constructed to be unable to degrade several amino acids. This strain was used as an expression host for the ...analysis of the amino acid configuration of nonribosomally synthesized peptides, including the novel peptide “xenotetrapeptide” from Xenorhabdus nematophila, by using a combination of labeling experiments and mass spectrometry. Additionally, the number of D‐amino acids in the produced peptide was assigned following simple cultivation of the expression strain in D2O.
No TATATATATA: An E. coli strain with deletions in five transaminases (ΔaspC ΔilvE ΔtyrB ΔavtA ΔybfQ) was unable to degrade several amino acids. This strain was used as expression host for the analysis of the amino acid configuration of nonribosomally synthesized peptides, including the novel “xenotetrapeptide” from Xenorhabdus nematophila.
The interaction in multisubunit non‐ribosomal peptide synthetases (NRPSs) is mediated by docking domains that ensure the correct subunit‐to‐subunit interaction. We introduced natural docking domains ...into the three‐module xefoampeptide synthetase (XfpS) to create two to three artificial NRPS XfpS subunits. The enzymatic performance of the split biosynthesis was measured by absolute quantification of the products by HPLC‐ESI‐MS. The connecting role of the docking domains was probed by deleting integral parts of them. The peptide production data was compared to soluble protein amounts of the NRPS using SDS‐PAGE. Reduced peptide synthesis was not a result of reduced soluble NRPS concentration but a consequence of the deletion of vital docking domain parts. Splitting the xefoampeptide biosynthesis polypeptide by introducing docking domains was feasible and resulted in higher amounts of product in one of the two tested split‐module cases compared to the full‐length wild‐type enzyme.
A breed apart: The non‐ribosomal peptide synthetase (NRPS) XfpS, which produces the peptides xefoampeptides A and B, can be artificially split without loss of biosynthetic activity by introducing docking domains into XfpS. In fact, in this case, the production of both peptides from the split system increased compared to product levels from the full‐length XfpS. Splitting XfpS into three subunits also led to a functional NRPS system.
Isovaleryl-CoA (IV-CoA) is usually derived from the degradation of leucine by using the Bkd (branched-chain keto acid dehydrogenase) complex. We have previously identified an alternative pathway for ...IV-CoA formation in myxobacteria that branches from the well-known mevalonate-dependent isoprenoid biosynthesis pathway. We identified 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (MvaS) to be involved in this pathway in Myxococcus xanthus, which is induced in mutants with impaired leucine degradation (e.g., bkd⁻) or during myxobacterial fruiting-body formation. Here, we show that the proteins required for leucine degradation are also involved in the alternative IV-CoA biosynthesis pathway through the efficient catalysis of the reverse reactions. Moreover, we conducted a global gene-expression experiment and compared vegetative wild-type cells with bkd mutants, and identified a five-gene operon that is highly up-regulated in bkd mutants and contains mvaS and other genes that are directly involved in the alternative pathway. Based on our experiments, we assigned roles to the genes required for the formation of IV-CoA from HMG-CoA. Additionally, several genes involved in outer-membrane biosynthesis and a plethora of genes encoding regulatory proteins were decreased in expression levels in the bkd⁻ mutant; this explains the complex phenotype of bkd mutants including a lack of adhesion in developmental submerse culture.
In higher plants, activating sequence-1 (as-1) of the cauliflower mosaic virus 35 S promoter mediates both salicylic acid (SA)- and auxin-inducible transcriptional activation. Originally found in ...promoters of several viral and bacterial plant pathogens, as-1-like elements are also functional elements of plant promoters activated in the course of a defense response upon pathogen attack. Nuclear as-1-binding factor (ASF-1) and cellular salicylic acid responseprotein (SARP) bind specifically to as-1. Four different tobacco bZIP transcription factors (TGA1a, PG13, TGA2.1, and TGA2.2) are potential components of either ASF-1 or SARP. Here we show that ASF-1 and SARP are very similar in their composition. TGA2.2 is a major component of either complex, as shown by supershift analysis and Western blot analysis of DNA affinity-purified SARP. Minor amounts of a protein immunologically related to TGA2.1 were detected, whereas TGA1a was not detectable. Overexpression of either TGA2.2 or a dominant negative TGA2.2 mutant affected both SA and auxin (2,4D) inducibility of various target promoters encoding as-1-like elements, albeit to different extents. This indicates that TGA2.2 is a component of the enhancosome assembling on these target promoters, both under elevated SA and 2,4D concentrations. However, the effect of altered TGA2.2 levels on gene expression was more pronounced upon SA treatment than upon 2,4D treatment.
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► The structure of bromoperoxidase II from the brown alga Ascophyllum nodosum was determined from a full length cloned cDNA. ► Bromoperoxidase II is a hexameric metalloprotein that ...binds per 67.4kDa-subunit 0.5 equivalents of vanadate. ► Vanadate is the cofactor for catalyzing oxidation of bromide by hydrogen peroxide. ► Enzymatic bromide oxidation furnishes a bromoelectrophile showing similar reactivity to molecular bromine. ► Bromoperoxidase II mimics biosynthesis of marine natural product methyl 4-bromopyrrole-2-carboxylate.
The sequence of bromoperoxidase II from the brown alga Ascophyllum nodosum was determined from a full length cloned cDNA, obtained from a tandem mass spectrometry RT-PCR-approach. The clone encodes a protein composed of 641 amino-acids, which provides a mature 67.4kDa-bromoperoxidase II-protein (620 amino-acids). Based on 43% sequence homology with the previously characterized bromoperoxidase I from A. nodosum, a tertiary structure was modeled for the bromoperoxidase II. The structural model was refined on the basis of results from gel filtration and vanadate-binding studies, showing that the bromoperoxidase II is a hexameric metalloprotein, which binds 0.5 equivalents of vanadate as cofactor per 67.4kDa-subunit, for catalyzing oxidation of bromide by hydrogen peroxide in a bi-bi-ping-pong mechanism (kcat=153s−1, 22°C, pH 5.9). Bromide thereby is converted into a bromoelectrophile of reactivity similar to molecular bromine, based on competition kinetic data on phenol bromination and correlation analysis. Reactivity provided by the bromoperoxidase II mimics biosynthesis of methyl 4-bromopyrrole-2-carboxylate, a natural product isolated from the marine sponge Axinella tenuidigitata.
Exchange of the native promoter to the arabinose‐inducible promoter PBAD was established in entomopathogenic bacteria to silence and/or activate gene clusters involved in natural product ...biosynthesis. This allowed the “on‐demand” production of GameXPeptides, xenoamicins, and the blue pigment indigoidine. The gene clusters for the novel “mevalagmapeptides” and the highly toxic xenorhabdins were identified by this approach.
Sweet induction: The promoters of several gene clusters involved in natural product biosynthesis were exchanged to the arabinose‐inducible PBAD promoter in entomopathogenic bacteria to result in “knock‐out” (without inducer) or tenfold overproduction (with the arabinose inducer), as shown for xenoamicin A (white crystals) and other compounds.
PoyD is a radical S‐adenosyl methionine epimerase that introduces multiple D‐configured amino acids at alternating positions into the highly complex marine peptides polytheonamide A and B. This novel ...post‐translational modification contributes to the ability of the polytheonamides to form unimolecular minimalistic ion channels and its cytotoxic activity at picomolar levels. Using a genome mining approach we have identified additional PoyD homologues in various bacteria. Three enzymes were expressed in E. coli with their cognate as well as engineered peptide precursors and shown to introduce diverse D‐amino acid patterns into all‐L peptides. The data reveal a family of architecturally and functionally distinct enzymes that exhibit high regioselectivity, substrate promiscuity, and irreversible action and thus provide attractive opportunities for peptide engineering.
A radical change: Radical S‐adenosyl methionine (SAM) epimerases is a new family of architecturally and functionally distinct bacterial enzymes. Three members from cyanobacteria were shown to introduce diverse D‐amino acid patterns into all‐L peptides. Their high regioselectivity, substrate promiscuity, and irreversible action provide interesting opportunities for peptide engineering.
Evolution-inspired engineering of nonribosomal peptide synthetases Bozhüyük, Kenan A J; Präve, Leonard; Kegler, Carsten ...
Science (American Association for the Advancement of Science),
2024-Mar-22, 2024-03-22, 20240322, Letnik:
383, Številka:
6689
Journal Article
Recenzirano
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.
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