How to tailor non-ribosomal peptide products—new clues about the structures and mechanisms of modifying enzymes (original) (raw)
Related papers
Nature Chemistry, 2019
Many important natural products are produced by non-ribosomal peptide synthetases (NRPSs) 1 .These giant enzyme machines activate amino acids in an assembly line fashion in which a set of catalytically active domains is responsible for the section, activation, covalent binding and connection of a specific amino acid to the growing peptide chain 1,2. Since NRPS are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would give access to a diverse range of peptides available biotechnologically. Here we describe a new fusion point inside condensation (C) domains of NRPSs that enables the efficient production of peptides, even containing non-natural amino acids, in yields higher than 280 mg/L. The technology called eXchange Unit 2.0 (XU2.0) also allows the generation of targeted peptide libraries and therefore might be suitable for the future identification of bioactive peptide derivatives for pharmaceutical and other applications.
Synthetic approaches to biologically active peptides and proteins including enzymes
Accounts of Chemical Research, 1989
career focused on the structure and function of enzymes and other bioiogically active peptides and proteins. He served on numerous scientific panels and advisory boards and also consulted extensively in industry. He was a member of the American Academy of Arts and Sciences and the National Academy of Sciences.
Amino Acids, 1996
The biosynthesis of microbial bioactive peptides is accomplished nonribosomally by large multifunctional enzymes consisting of linearly arranged building blocks of 1,000-1,500 amino acid residues. Each of these units acts as an independent enzyme which catalyzes the selection, activation, and in some cases modification (epimerization, N-methylation) of its cognate amino acid, as well as the elongation of the peptide product. The specific linkage of amino acid activating modules upon such polyenzymes defines the sequence of the peptide product. A series of functional domains could be identified upon an amino acid activating module which are involved in the sequential reactions in nonribosomal peptide biosynthesis.
Recent Advances in Enzyme Engineering through Incorporation of Unnatural Amino Acids
Biotechnology and Bioprocess Engineering, 2019
The development of new enzyme engineering technologies has been actively pursued as the industrial use of biocatalysts is rapidly increasing. Traditional enzyme engineering has been limited to changing the functional properties of enzymes by replacing one amino acid with the other 19 natural amino acids. However, the incorporation of unnatural amino acids (UAAs) has been exploited to manipulate efficient enzymes for biocatalysis. This has been an effective enzyme engineering technique by complementing and extending the limits of traditional enzymatic functional changes. This review paper describes the basic functions of the new functional groups of UAAs used in enzyme engineering and the utilization of UAAs in the formation of chemical bonds in the proteins. The recent developments of UAA-mediated enzymology and its applicability in industry, pharmaceutical and other research areas to overcome the limitations of existing enzymes is also emphasized.
Chemistry - A European Journal, 2013
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with a high degree of structural diversity and a wide variety of bioactivities. Understanding the biosynthetic machinery of these RiPPs will benefit the discovery and development of new molecules with potential pharmaceutical applications. In this Concept article, we discuss the features of the biosynthetic pathways to different RiPP classes, and propose mechanisms regarding recognition of the precursor peptide by the post-translational modification enzymes. We propose that the leader peptides function as allosteric regulators that bind the active form of the biosynthetic enzymes in a conformational selection process. We also speculate how enzymes that generate polycyclic products of defined topologies may have been selected for during evolution.
Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins
Nature Chemistry, 2016
O ver 100 years have passed since the first attempts to form amide bonds using simple starting materials. Despite this, great efforts are still being invested to develop new methods of efficiently forming amide bonds at low cost using environmentally friendly reagents 1. Synthesis of short peptide sequences (20-40 amino acids (AA)) is considered a relatively straightforward process with some exceptions such as amyloid β-peptides, transmembrane peptides and amphiphilic peptides. These difficult peptide sequences require special coupling reagents and synthetic strategies to achieve efficient synthesis. The relative ease by which essentially any short tailored peptide can be synthesized by applying solid-phase peptide synthesis (SPPS) has arisen from the continuous development of a wide variety of excellent coupling reagents, resins with better physical properties, novel linkers and orthogonal protecting groups 2. Improvement in chromatographic methods and analytical instrumentation for purification and characterization has also simplified the process of synthesizing peptides. The high efficiency of peptide synthesis coupled with automation has greatly expanded the use of peptides in several research areas, such as nanotechnology, drug discovery and total chemical synthesis of proteins. Chemical protein synthesis, which relies on chemoselective peptide ligation methods, such as native chemical ligation (NCL) 3 , offers complete control of the atomic composition of the polypeptide sequence, making it advantageous over molecular biology approaches in some areas of research (Fig. 1) 4. For example, it enables the incorporation of post-translational modifications (PTMs) into proteins with high homogeneity and in usable quantities, which can enable the role of these modifications on the protein structure and function to be examined. This method for the production of posttranslationally modified proteins has generated immense interests among many groups who have prepared, for example, glycosylated, phosphorylated and ubiquitinated proteins that have enabled otherwise difficult studies 5. Another exciting aspect of chemical protein synthesis is the ability to prepare mirror-image proteins, which are increasingly finding impressive applications in drug discovery 6 and protein crystallography 7 .
bioRxiv (Cold Spring Harbor Laboratory), 2023
Non-ribosomal peptides are a diverse and medically important group of natural products. They are biosynthesised by modular non-ribosomal peptide synthetase (NRPS) assembly-lines in which domains from each module act in concert to incorporate a specific amino acid into a peptide. This modular biosynthesis has driven efforts to generate new peptide analogues by substituting amino acid specifying domains. Rational NRPS engineering has increasingly focused on using evolutionarily favoured recombination sites for domain substitution. Here, we present an alternative approach inspired by evolution, which involves large-scale diversification and screening. By adopting a metagenomic approach of amplifying amino acid specifying domains from metagenomic DNA derived from soil, we were able to substitute over 1,000 unique domains into a pyoverdine NRPS. To identify functional domain substitutions, we employed fluorescence and mass spectrometry screening techniques, followed by sequencing. This comprehensive screening process successfully identified more than 100 functional domain substitutions, resulting in the production of 16 distinct pyoverdines as major products. The significance of this metagenomic approach lies in its ability to shift the focus of engineering non-ribosomal peptide biosynthesis. Instead of relying on a high success rate of individual domain substitution, we have developed effective methods that enable the exploration of a broader range of substitutions. This opens new possibilities for the discovery and production of novel non-ribosomal peptides with diverse biological activities.
Peptide synthesis: chemical or enzymatic
Electronic Journal of Biotechnology, 2007
CD: circular dichroism CLEC: cross linked enzyme crystals DDC: double dimer constructs ESI: electrospray ionization HOBT: hydroxybenzotriazole HPLC: high performance liquid hromatography KCS: kinetically controlled synthesis MALDI: matrix-assisted laser desorption ionization MAP: multiple antigen peptide system MS: mass spectrometry NMR: nuclear magnetic resonance SPS: solution phase synthesis SPPS: solid-phase peptide synthesis t-Boc: tert-butoxycarbonyl TCS: thermodynamically controlled synthesis TFA: trifluoroacetic acid Peptides are molecules of paramount importance in the fields of health care and nutrition. Several technologies for their production are now available, among which chemical and enzymatic synthesis are especially relevant. The present review pretends to establish a non-biased appreciation of the advantages, potentials, drawbacks and limitations of both technologies. Chemical synthesis is thoroughly reviewed and their potentials and limitations assessed, focusing on the different strategies and challenges for large-scale synthesis. Then, the enzymatic synthesis of peptides with proteolytic enzymes is reviewed considering *Corresponding author medium, biocatalyst and substrate engineering, and recent advances and challenges in the field are analyzed. Even though chemical synthesis is the most mature technology for peptide synthesis, lack of specificity and environmental burden are severe drawbacks that can in principle be successfully overcame by enzyme biocatalysis. However, productivity of enzymatic synthesis is lower, costs of biocatalysts are usually high and no protocols exist for its validation and scale-up, representing challenges that are being actively confronted by intense research and development in this area. The combination of chemical and enzymatic JAKUBKE, Hans-Dieter; KUHL, Peter and KONNECKE, Andreas. Basic principles of protease-catalized peptide bond formation. Insights into the mechanism and catalysis of the native chemical ligation reaction. MOZAHEV, Vadim V.; BELOVA, Alla B.; SERGEEVA, María V. and MARTINEK, Karel. Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis.