Discovery, Characterisation and Engineering of Non-Ribosomal Peptide Synthetases and Phosphopantetheinyl Transferase Enzymes PDF Download
Are you looking for read ebook online? Search for your book and save it on your Kindle device, PC, phones or tablets. Download Discovery, Characterisation and Engineering of Non-Ribosomal Peptide Synthetases and Phosphopantetheinyl Transferase Enzymes PDF full book. Access full book title Discovery, Characterisation and Engineering of Non-Ribosomal Peptide Synthetases and Phosphopantetheinyl Transferase Enzymes by Katherine Robins. Download full books in PDF and EPUB format.
Author: Katherine Robins
Publisher:
ISBN:
Category : Enzymes
Languages : en
Pages : 0
Get Book Here
Book Description
Author: Katherine Robins
Publisher:
ISBN:
Category : Enzymes
Languages : en
Pages : 0
Get Book Here
Book Description
Author: Michael Burkart
Publisher: Springer Nature
ISBN: 1071632140
Category : Science
Languages : en
Pages : 324
Get Book Here
Book Description
This volume provides new technologies on NRPSs and related carrier protein dependent synthases, including polyketide synthases (PKS) and fatty acid synthases (FAS). Chapters detail enzymology, structural biology, proteopromics, chemical biology, natural product chemistry, and bioinformatics. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and methods, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, Non-Ribosomal Peptide Biosynthesis and Engineering: Methods and Protocols aims to feature methods that will be beneficial to new researchers, and those wanting to adopt new methodologies into their research.
Author: Bradley S. Evans
Publisher:
ISBN:
Category :
Languages : en
Pages :
Get Book Here
Book Description
Nonribosomal peptides are an important class of natural products including the life-saving antibiotics penicillin and vancomycin and the deadly microcystins and cereulide. This important category of natural products is thus a focus of many research labs around the world, including the Kelleher lab. These often complex peptides are assembled by large enzymes having multiple active sites for activation, peptide bond formation as well as chemical tailoring. The peptides are assembled on nonribosomal peptide synthetase (NRPS) assembly lines covalently tethered to the synthetases throughout biosynthesis. Mass spectrometry is an especially well suited tool for analyzing both the products of NRPS pathways and the machinery that produces them. A review of NRPS enzymology and contemporary mass spectrometric methods for their analysis is given in Chapter 1. The work described in Chapter 2 for the first time brings to bear the power of mass spectrometry and directed evolution against the challenge of reengineering NRPSs. A collaborative effort between members of the Kelleher lab that led to the development of a natural product discovery platform based upon proteomic analysis is described in Chapter 3. The first discovery, sequencing and characterization of a novel NRPS biosynthetic gene cluster using this new proteomics based platform in the Kelleher lab is described in Chapter 4. In summary, this thesis is founded on and extends upon the pioneering work of in vitro characterization of NRPSs performed in the Kelleher lab over the last decade and paves the way for an explosion of future discoveries using these and yet to be developed techniques.
Author: Philipp Stephan
Publisher:
ISBN:
Category :
Languages : de
Pages : 0
Get Book Here
Book Description
Over the last decades, discovery rates of new antibiotics have declined while the occurrence of multi-resistant pathogens has increased, leading towards a post-antibiotic era. Many current antibiotics are natural products or derivatives, known for their excellent bioactivities but complex structures that complicate synthetic production. Many antibiotics, like penicillin and vancomycin, are peptides produced by nonribosomal peptide synthetases (NRPS). These multi-modular enzyme complexes function like assembly lines, with each module adding a specific amino acid to the peptide through the biocatalytic activities of distinct domains. Targeted NRPS engineering to alter structures of the produced peptides has faced challenges, but directed evolution of NRPS domains seems to offer a promising solution. This thesis presents a straightforward method for the directed evolution of adenylation (A) domains using LC-MS/MS detection of dipeptides in cell lysates, demonstrating its power by engineering the GrsB1 A domain from gramicidin S biosynthesis to select for L-piperazic acid (Piz) instead of its native substrate L-Pro without reducing enzyme activity. The modified antibiotic, Piz-gramicidin S, showed improved bioactivity. Despite compatibility issues between NRPS domains, kinetic analyses revealed that A domain substrate preferences ultimately determine final product ratios. Additionally, the thesis proposes a novel DNA-based approach for NRPS engineering, using zinc finger domains that bind specific DNA sequences to simplify module rearrangement by making NRPSs DNA-templated. This method offers a more efficient alternative to traditional cloning techniques by replacing the handling of large NRPS genes with much shorter sequences of the zinc finger domains. Overall, this research advances the engineering of NRPSs for developing new antibiotics with better efficacy against resistant pathogens and highlights the usefulness of directed evolution in this context.
Author: Shayhan Chunkath
Publisher:
ISBN:
Category : Enzymes
Languages : en
Pages : 111
Get Book Here
Book Description
Non-ribosomal Peptide Synthetases (NRPSs) are large, modular, multi domain enzymes that function to synthesize a specific short peptide product in an assembly-line fashion. This is accomplished by the selection, loading and linking of amino or carboxylic acid monomers via peptide bonding. Many NRPS enzymes have been identified and studied in both bacterial and fungal genomes, and allow for the production of secondary metabolites. The ruminant methanogen, Methanobrevibacter ruminantium M1, has recently been shown to possess two NRPS encoding genes, representing the first to be discovered in Archaea. The non-ribosomal peptide products produced by the methanogen NRPS enzymes have not been identified. I have conducted research on one of these methanogen NRPS enzymes, mru_0351. Recombinant expression of both a single domain (mru_0351-A3L) and a multi domain (mru_0351-C2R) protein construct excised from the mru_0351 NRPS has allowed for their characterisation using a host of biochemical and biophysical techniques. Efforts have focused on interrogating the reactions catalysed by the NRPS adenylation domains, which are responsible for initial substrate selection, and thus serve a key gatekeeping role in committing a given substrate to the biosynthetic pathway. Adenylation domains sequentially catalyse two reactions: adenylation and thiolation. The adenylation reaction serves as a substrate filtering step, electively activating cognate monomers. The thiolation reaction then covalently tethers the activated substrate to a post-translationally attached 4'-phosphopantetheine group on the NRPS assembly. Crystallographic and small angle X-ray scattering (SAXS) techniques were used to provide structural insights into the mechanics of the methanogen mru_0351 NRPS. Diffracting protein crystals of proteolytic products from mru_0351-C2R and mru_0351-A3L were produced, indicating promising leads towards structural determination of these NRPS constructs. SAXS analysis revealed subtle conformational changes in the multidomain structure of mru_0351-C2R upon addition of substrates. Development of mass spectrometry methods to identify intermediates tethered to the 4'-phosphopantetheinyl post-translational modification revealed that the mru_0351 NRPS enzyme's adenylation domains confer additional selectivity via the thiolation reaction. Thiolation reaction substrate preference has not previously been reported in NRPS enzymes, and this discovery may have broad implications for how module specificity is conferred in NRPS enzymes in general.
Author: Jennifer Fick Brannock
Publisher:
ISBN:
Category :
Languages : en
Pages : 184
Get Book Here
Book Description
Author: Amanda Jo Platt
Publisher:
ISBN:
Category : Biochemistry
Languages : en
Pages : 0
Get Book Here
Book Description
Non-ribosomal peptide synthetases (NRPSs) are responsible for full or partial biosynthesis of medically invaluable compounds including antifungals, immunosuppressants, and antibiotics like vancomycin. Synthetases have a modular architecture and function in an assembly line manner where peptide products are extended and modified as they are passed between modules. Engineering synthetases by replacing or re-ordering modules is an attractive strategy to generate "unnatural natural products". However, successful combinatorial engineering requires characterization of new NRPS domains and an improved understanding of the protein-protein interactions that govern synthetase function. This work focused on determining domain composition and substrate specificity of three-domain synthetases from bacterial species Lactobacillus iners and Scytonema hofmannii. Sequence analysis predicted the bacterial synthetases to contain adenylation- and carrier protein-domains, as well as C-terminal domains with hypothetical functions. We adopted a recently published mass spectrometry method to probe substrate specificity of these A-domains and they were shown to activate proline and glycine. Sequence homology between bacterial three-domain NRPSs and the Drosophila melanogaster Ebony synthetase, led us to interrogate Ebony in more detail and we focused on protein-protein interactions between domains within the synthetase. Dissection of the synthetase into separate proteins showed that certain combinations failed to produce product, but nine percent of activity was preserved following dissection of Ebony into a di-domain with detached C-terminal domain. Collectively, this work contributes to NRPS engineering efforts by characterizing the specificity of new domains and developing a platform to assess activity of a dissected synthetase. This information can be leveraged to utilize three-domain synthetases as engineering tools to characterize additional domains and generate new dipeptides.
Author: Andreas Tietze
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
Get Book Here
Book Description
Author: Jeremy George Owen
Publisher:
ISBN:
Category : Enzymes
Languages : en
Pages : 243
Get Book Here
Book Description
Author: Reto Daniel Zwahlen
Publisher:
ISBN: 9789403406732
Category :
Languages : en
Pages :
Get Book Here
Book Description
