Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines (original) (raw)
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Microbiology (Reading, England), 2004
Nonribosomal peptide synthetases (NRPSs) are giant multi-domain enzymes that catalyse the biosynthesis of many commercially important peptides produced by bacteria and fungi. Several studies over the last decade have shown that many of the individual domains within NRPSs exhibit significant substrate selectivity, which impacts on our ability to engineer NRPSs to produce new bioactive microbial peptides. Adenylation domains appear to be the primary determinants of substrate selectivity in NRPSs. Much progress has been made towards an empirical understanding of substrate selection by these domains over the last 5 years, but the molecular basis of substrate selectivity in these domains is not yet well understood. Perhaps surprisingly, condensation domains have also been reported to exhibit moderate to high substrate selectivity, although the generality of this observation and its potential impact on engineered biosynthesis experiments has yet to be fully elucidated. The situation is le...
Evolution-guided engineering of nonribosomal peptide synthetase adenylation domains
Chem. Sci., 2013
Hormaomycin is a structurally unusual morphogenic and antibiotic peptide biosynthesized by a bacterial nonribosomal peptide synthetase (NRPS). Bioinformatic analysis suggested that parts of the NRPS adenylation (A) domains had recombined during evolution, resulting in a major switch of substrate specificity. This feature inspired us to create A domains with altered substrates based on the putative recombination points. Following characterization of all native hormaomycin A domains, engineered versions were constructed and characterized. Three of the enzymes displayed an almost identical specificity profile to that of native domains recognizing the same substrates. The data support the evolutionary hypothesis regarding the emergence of the hormaomycin pathway and suggest new strategies in NRPS engineering. ; Fax: +41 44 6321378; Tel: +41 44 6339443 †Electronic supplementary information (ESI) available. See
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.
Molecular BioSystems, 2008
Non-ribosomal peptide products often contain modified building blocks or postassembly line alterations of their peptide scaffolds with some of them being crucial for biological activity. These reactions such as halogenation, hydroxylation or glycosylation are mostly catalyzed by individual enzymes associated with the respective biosynthesis cluster. The versatile nature of these chemical modifications gives rise to a high degree of structural and functional diversity. Recent progress in this area enhances our insight about the mechanisms of these enzymes. Biotechnological applications might include the synthesis of novel, non-ribosomal peptide products or modified amino acid building blocks for pharmaceutical research.
NRPS toolbox for the discovery of new nonribosomal peptides and synthetases
Nonribosomal peptide synthetases are huge multi-enzymatic complexes synthesizing peptides, but not through the classical process of transcription and then translation. The synthetases are organised in modules, each one integrating an amino acid in the final peptide. The modules are divided in domains providing specialized activities. So, those enzymes are as diverse as their products. We present our toolbox designed to annotate them accurately and promising results obtained on some Burkholderia, Bacillus and Pseudomonas genomes.
The Multiple Carrier Model of Nonribosomal Peptide Biosynthesis at Modular Multienzymatic Templates
Journal of Biological Chemistry, 1996
Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a "multiple carrier model" of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.