Dirk Schwarzer

Chemoselective Ligation and Modification Strategies for Peptides and Proteins

The investigation of biological processes by chemical methods, commonly referred to as chemical biology, often requires chemical access to biologically relevant macromolecules such as peptides and proteins. Building upon solid-phase peptide synthesis, investigations have focused on the development of chemoselective ligation and modification strategies to link synthetic peptides or other functional units to larger synthetic and biologically relevant macromolecules. This Review summarizes recent developments in the field of chemoselective ligation and modification strategies and illustrates their application, with examples ranging from the total synthesis of proteins to the semisynthesis of naturally modified proteins.

Nonribosomal peptides: from genes to products

The ability to synthesize nonribosomally small bioactive peptides that find application in modern medicine is widely spread among microorganisms. As broad as the spectrum of biological activities is the structural diversity of these peptides, which are mostly cyclic or branched cyclic compounds containing non-proteinogenic amino acids, small heterocyclic rings and other unusual modifications in the peptide backbone. They are synthesized by multimodular enzymes, the so-called nonribosomal peptide synthetases (NRPSs), from simple building blocks. Biochemical and genetic studies have unveiled the key principles of nonribosomal peptide syntheses, as well as the realization of many structural features of these peptides. This review focuses on recent results in NRPS research and highlights how this knowledge can be exploited for biotechnological purposes. In addition, possibilities and limitations for prediction of structural features of uncharacterized NRPSs and approaches for their engineering are discussed.

Ways of Assembling Complex Natural Products on Modular Nonribosomal Peptide Synthetases

Nonribosomal peptide synthetases (NRPSs) catalyze the assembly of a large number of complex peptide natural products, many of which display therapeutically useful activity. Each cycle of chain extension is carried out by a dedicated module of the multifunctional enzymes. A module harbors all the catalytic units, which are referred to as domains, necessary for recognition, activation, covalent binding, and optionally modification of a single building block monomer, as well as for peptide‐bond formation with the growing chain. A terminal domain releases the full‐length peptide chain from the enzyme complex. Recent characterization of many NRPS systems revealed several examples where the sequence of the product does not directly correspond to the linear arrangement of modules and domains within the enzyme(s). It is now obvious that these systems cannot be regarded as rare exceptions of the common NRPS architecture but rather represent more complicated variations of the NRPS repertoire to increase their biosynthetic potential. In most of these cases unusual peptide structures of the products are observed, such as structures with side‐chain acylation, cyclization involving the peptide backbone and/or side chains, and transfer of the peptide chain onto soluble small‐molecule substrates. These findings indicate a previously unexpected higher versatility of the modules and domains in terms of both catalytic potential and interaction within the multifunctional protein templates. We propose to classify the known NRPS systems into three groups, linear NRPSs (type A), iterative NRPSs (type B), and nonlinear NRPSs (type C), according to their biosynthetic logic. Understanding the various biosynthetic strategies of NRPSs will be crucial to fully explore their potential for engineered combinatorial biosynthesis.

Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases

Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme. A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation. However, the actual role of TEIIs in nonribosomal peptide synthesis remained obscure. Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin (TEIIsrf) and bacitracin (TEIIbac). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4′-phosphopantetheine (4′PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs. For TEIIsrf, a KM of 0.9 μM and a kcat of 95 min−1 was determined for acetyl-PCP hydrolysis. Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of nonribosomal peptide synthesis. However, this reaction is unlikely to be of physiological relevance. Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid synthesis were also not significantly hydrolyzed, as investigated with TEIIsrf. These findings support a model in which the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4′PP transferases that use not only free CoA, but also acyl-CoAs as 4′PP donors.

Multimodular biocatalysts for natural product assembly

Abstract. Nonribosomal peptides and polyketides represent a large class of natural products that show an extreme structural diversity and broad pharmacological relevance. They are synthesized from simple building blocks such as amino or carboxy acids and malonate derivatives on multimodular enzymes called nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), respectively. Although utilizing different substrates, NRPSs and PKSs show striking similarities in the modular architecture of their catalytic domains and product assembly-line mechanism. Among these compounds are well known antibiotics (penicillin, vancomycin and erythromycin) as well as potent immunosuppressive agents (cyclosporin, rapamycin and FK 506). This review focuses on the modular organization of NRPSs, PKSs and mixed NRPS/PKS systems and how modules and domains that build up the biosynthetic templates can be exploited for the rational design of recombinant enzymes capable of synthesizing novel compounds.

Collisional deactivation of vibrationally highly excited azulene in compressed liquids and supercritical fluids

The collisional deactivation of vibrationally highly excited azulene was studied from the gas to the compressed liquid phase. Employing supercritical fluids like He, Xe, CO2, and ethane at pressures of 6–4000 bar and temperatures ≥380 K, measurements over the complete gas–liquid transition were performed. Azulene with an energy of 18 000 cm−1 was generated by laser excitation into the S1 and internal conversion to the S0*‐ground state. The subsequent loss of vibrational energy was monitored by transient absorption at the red edge of the S3←S0 absorption band near 290 nm. Transient signals were converted into energy‐time profiles using hot band absorption coefficients from shock wave experiments for calibration and accounting for solvent shifts of the spectra. Under all conditions, the decays were monoexponential. At densities below 1 mol/l, collisional deactivation rates increased linearly with fluid density. Average energies 〈ΔE〉 transferred per collision agreed with data from dilute gas phase experiment...

Ebony, a novel nonribosomal peptide synthetase for β-alanine conjugation with biogenic amines in Drosophila

Using Ebony protein either expressed in Escherichia coli or in Schneider S2 cells, we provide evidence for its substrate specificity and reaction mechanism. Ebony activates β-alanine to aminoacyladenylate by an adenylation domain and covalently attaches it as a thioester to a thiolation domain in a nonribosomal peptide synthetase (NRPS) related mechanism. In a second reaction, biogenic amines act as external nucleophiles on β-alanyl-S-pantetheine-Ebony, thereby releasing in a fast reaction the dipeptide (peptidoamine) in a process that is novel in higher eucaryotes. Therefore, we define Ebony as a β-alanyl-biogenic amine synthetase. Insight into the reaction mechanism stems from mutational analysis of an invariant serine that disclosed Ebony as a multienzyme with functional analogy to the starting modules of NRPSs. In light of a putative biogenic amine-deactivating capacity, Ebony function in the nervous system must be reconsidered. We propose that in the Drosophila eye Ebony is involved in the transmission process by inactivation of histamine through β-alanyl conjugation.

Chromatin regulated interchange between polycomb repressive complex 2 (PRC2)-Ezh2 and PRC2-Ezh1 complexes controls myogenin activation in skeletal muscle cells

BackgroundPolycomb group (PcG) genes code for chromatin multiprotein complexes that are responsible for maintaining gene silencing of transcriptional programs during differentiation and in adult tissues. Despite the large amount of information on PcG function during development and cell identity homeostasis, little is known regarding the dynamics of PcG complexes and their role during terminal differentiation.ResultsWe show that two distinct polycomb repressive complex (PRC)2 complexes contribute to skeletal muscle cell differentiation: the PRC2-Ezh2 complex, which is bound to the myogenin (MyoG) promoter and muscle creatine kinase (mCK) enhancer in proliferating myoblasts, and the PRC2-Ezh1 complex, which replaces PRC2-Ezh2 on MyoG promoter in post-mitotic myotubes. Interestingly, the opposing dynamics of PRC2-Ezh2 and PRC2-Ezh1 at these muscle regulatory regions is differentially regulated at the chromatin level by Msk1 dependent methyl/phospho switch mechanism involving phosphorylation of serine 28 of the H3 histone (H3S28ph). While Msk1/H3S28ph is critical for the displacement of the PRC2-Ezh2 complex, this pathway does not influence the binding of PRC2-Ezh1 on the chromatin. Importantly, depletion of Ezh1 impairs muscle differentiation and the chromatin recruitment of MyoD to the MyoG promoter in differentiating myotubes. We propose that PRC2-Ezh1 is necessary for controlling the proper timing of MyoG transcriptional activation and thus, in contrast to PRC2-Ezh2, is required for myogenic differentiation.ConclusionsOur data reveal another important layer of epigenetic control orchestrating skeletal muscle cell terminal differentiation, and introduce a novel function of the PRC2-Ezh1 complex in promoter setting.

Viscosity and solvent dependence of low-barrier processes : photoisomerization of cis-stilbene in compressed liquid solvents

The photoisomerization of cis‐stilbene in liquid solution was studied by time‐resolved excited‐state absorption spectroscopy using 306 nm pump and 612 nm probe pulses of 100 fs width. Transient absorption signals were found to decrease exponentially with time. Decay rate constants were determined over the pressure range 1–4000 bars at temperatures of 295 and 390 K in a series of alkane solvents as well as in methanol, acetonitrile, and in polymethylmethacrylate (PMMA). The viscosity dependence confirms the existence of two pathways of the reaction, one leading to ground‐state cis‐ and trans‐stilbene (C/TS), the other to dihydrophenanthrene (DHP). Whereas the DHP component shows only little viscosity dependence up to 4 kbar, the C/TS component is characterized by rate constants which are inversely proportional to the solvent viscosity. This is in contrast to earlier conclusions from studies with solvents at 1 bar. The C/TS process shows practically no temperature dependence apart from that of the viscosity...

Cluster and barrier effects in the temperature and pressure dependence of the photoisomerization of trans‐stilbene

The pressure and temperature dependence of the photoisomerization rate coefficient of trans‐stilbene in the S1 state have been measured in the solvents C2H6, C3H8, C4H10, Xe, Co2, SF6, and CHF3. At constant temperature, the pressure dependences up to 6 kbar can be well represented by the Kramers–Smoluchowski model. The comparison of results in different solvents clearly indicates the importance of reactant–solvent cluster formation modifying the height and imaginary frequency of the barrier. The change of the temperature dependence with pressure points towards a multidimensional barrier of nonseparable character. Multidimensional barrier effects manifest themselves most clearly via the temperature dependence of the rate coefficient in the Kramers–Smoluchowski limit.