Characterization of Adenylating Enzymes and Domain Interactions Involved in Insect and Bacterial Non-ribosomal Peptide Synthesis PDF Download
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Author: Amanda Jo Platt
Publisher:
ISBN:
Category : Biochemistry
Languages : en
Pages : 0
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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: Amanda Jo Platt
Publisher:
ISBN:
Category : Biochemistry
Languages : en
Pages : 0
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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: Aleksa Stanišić
Publisher:
ISBN:
Category :
Languages : de
Pages :
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Book Description
Considering the ongoing rise of the multidrug-resistant bacterial infections, it is essential to expand the available repertoire of therapeutic agents. Microbial natural products are an indispensable source of novel activities and continue to serve as our main provider of antibiotics and chemotherapeutics. Nonribosomal peptides are among the most widespread natural products in bacteria and fungi. Their importance is best illustrated by their complexity and the amounts of resources dedicated to building the underlying biosynthetic machineries nonribosomal peptide synthetases (NRPS). These gigantic, multidomain enzymes synthesize peptides by linking individual amino acid units in an assembly line fashion. Six decades of NRPS research have resulted in several remarkable tailoring successes. However, the lack of mechanistic understanding of the inner workings of NRPSs has prevented the development of a general workflow which would reliably generate functional enzymes and new drugs. Aspiring to alleviate these obstacles, this thesis offers critical insights into adenylation and the interplay with condensation, two fundamental NRPS reactions.
Author: Michael Andrew Fischbach
Publisher:
ISBN:
Category : Biomolecules
Languages : en
Pages : 264
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Book Description
Author: Clarisse Chiche-Lapierre
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
"The fascinating microscopic world of bacteria and fungi is rich in small bioactive compounds produced by the organisms in their constant war of survival. Among this treasure trove of compounds were found many of the pharmaceutical compounds invaluable to humankind. One such class of compounds is the nonribosomal peptides (NRPs) produced by massive multimodular enzymes called nonribosomal peptide synthetases (NRPSs). These machines consist of a large number of catalytic domains operating in modules, where each module is responsible for the addition of one amino acid building block in the growing peptide chain of the final product. The adenylation domains (A domains) are responsible for selecting one specific residue and loading it onto the prosthetic arm of the peptidyl carrier protein domains (PCP domains) shuttling the growing chain along the NRPS assembly line. One such NRPS is found in Bacillus stratosphericus and is responsible for the production of a compound similar to valinomycin and cereulide, two very powerful toxins. The initial module contains an atypical domain, the ketoreductase domain (Kr domain). To shed light on how the structural elements of the synthetase work together, we embarked on solving the protein structure of parts of this initiation module, the A-Kr-PCP, through X-ray crystallography.The crystallography of A-Kr-PCP proved to be very challenging, but three factors made it possible for us to solve: controlling the presence of the prosthetic arm attached to the PCP domain and what substrate is bound to it; the use of advanced techniques of cryoprotection and dehydration of the crystals, and finally, the process of iterative omit-map sharpening combined with four-fold non-crystallographic symmetry averaging. This work resulted in two individual protein structures: the StsA_A-KrT initiation module solved to a resolution of 3.4-3.9Å and the StsA A domain solved to a resolution of 2.3 Å. The structure of StsA_AKrT contains three distinctly novel features. It contains the first structure of an NRPS Kr domain. It is also the first structure of a Kr-containing NRPS module, allowing us to visualize how the Kr domain is embedded into the synthetase. Finally, the structure contains the first structure of a ketoacid-selecting A domain. Contrary to expectations, the A domain is uninterrupted by the Kr domain, and the two domains show very little surface contact. The structure also revealed a surprising feature: a novel pseudo Asub (pAsub) domain, interrupted by the A-Kr di-domain, dividing it into two parts located over 1000 residues apart in the primary sequence, brought together through a 39-amino-acid long strand swap between dimeric partners, seemingly inducing dimerization. The dimer:monomer ratio can be controlled to some extent by controlling the length of the linker region involved in the domain swap. The pAsub domain has the same structure as the canonical Asub domain but holds no catalytic function. The module is able to fold properly and adenylate its substrate without this curious subdomain. The keto-acid-selecting A domain has the same overall structure as amino-acid-selecting ones, but some of the highly conserved motifs and substrate binding residues differ. The [alpha]-keto substrate seems to be accommodated by the mutation in StsA of a proline to a methionine residue, whose backbone carbonyl may rotate 40° to interact with the [alpha]-carbonyl group of the substrate through a reciprocal carbonyl-carbonyl interaction. We showed that the reverse mutation allowed for an amino acid selecting A domain from linear gramicidin synthetase A to select and adenylate the [alpha]-keto version of its cognate substrate. The Kr domain has the same overall structure as those found in other types of synthesis systems, and seem to function through a similar mechanism. Finally, we propose a design for inhibitors that would trap the Kr domain in the substrate binding and ketoreduction state"--
Author: Katherine Robins
Publisher:
ISBN:
Category : Enzymes
Languages : en
Pages : 194
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Book Description
Non-ribosomal peptide synthetases (NRPSs) are multi-modular biosynthetic enzymes that are responsible for the production of many bioactive secondary metabolites produced by microorganisms. They are activated by phosphopantetheinyl transferase (PPTase) enzymes, which attach an essential prosthetic group to a specific site within a “carrier protein” (CP) domain that is an integral part of each NRPS module. Of particular importance in this work is the NRPS BpsA, which produces a blue pigment called indigoidine; but only when BpsA has first been activated by a PPTase. BpsA can be used as a reporter for PPTase activity, to identify PPTases and/or measure their activity. Several CP-substituted BpsA variants were used, in order to study and identify PPTases which may recognise different CP domains. The first part of the research described in this thesis examined the features of foreign CP interactions within BpsA that made these functional substitutions possible. Two key residues, the +4 and +24 positions relative to an invariant serine, were found to be highly important; with appropriate substitutions at these positions yielding active CP-substituted variants. Wild type BpsA and the CP-substituted variants were then used as the basis of a screen to discover new PPTase genes, and associated natural product biosynthetic genes, from metagenomic libraries. The vast majority of bacteria that produce bioactive secondary metabolites are unable to be cultured under laboratory conditions; screening metagenomic libraries is a way to access this untapped biodiversity in order to discover new natural products. Two environmental DNA libraries were screened, and PPTase genes were identified via their ability to activate BpsA, giving rise to blue colonies in high throughput agar plate screens. This screen proved to be a powerful enrichment strategy with almost half of the novel 21 PPTase genes recovered also linked to biosynthetic gene clusters.
Author: Derek Barton
Publisher: Newnes
ISBN: 0080912834
Category : Science
Languages : en
Pages : 11380
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Book Description
Comprehensive Natural Products Chemistry
Author: Christopher T. Walsh
Publisher: Royal Society of Chemistry
ISBN: 1788010760
Category : Science
Languages : en
Pages : 787
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Book Description
This textbook describes the types of natural products, the biosynthetic pathways that enable the production of these molecules, and an update on the discovery of novel products in the post-genomic era.
Author: Dieter Söll
Publisher:
ISBN: 9781555818333
Category : Aminoacyl-tRNA synthetases
Languages : en
Pages : 572
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Book Description
Author: P.A.H.M. Bakker
Publisher: Springer Science & Business Media
ISBN: 1402067763
Category : Science
Languages : en
Pages : 127
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Book Description
In the context of increasing concern for food and environmental quality, use of Plant Growth-Promoting Rhizobacteria (PGPR) for reducing chemical inputs in agriculture is a potentially important issue. This book provides an update by renowned international experts on the most recent advances in the ecology of these important bacteria, the application of innovative methodologies for their study, their interaction with the host plant, and their potential application in agriculture.
Author: Nancy P. Keller
Publisher: Humana Press
ISBN: 9781627031219
Category : Science
Languages : en
Pages : 288
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Book Description
Filamentous fungi have long been known for their ability to produce an enormous range of unusual chemical compounds known as secondary metabolites, many of which have potentially useful antibiotic or pharmacological properties. Recent focus on fungal genomics coupled with advances in detection and molecular manipulation techniques has galvanized a revitalization of this field. Fungal Secondary Metabolism: Methods and Protocols is aimed at providing the key methodologies currently in use and necessary for accessing and exploiting the natural product information provided by the genomes of this large and varied kingdom. Written by active researchers in the field, the chapters deal with all the steps necessary, from optimization of fungal culture conditions for metabolite production, through rapid genome sequencing and bioinformatics, and genetic manipulations for functional analysis, to detection and testing of metabolites. In addition, chapters on basic science address approaches to the genetic regulation, protein biochemistry, and cellular localization of the biosynthetic pathways. Written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Practical and hands-on, Fungal Secondary Metabolism: Methods and Protocols encourages new investigators to enter the field and expands upon the expertise and range of skills of those already researching fungal natural products.
