Springer protocols feed by Protein Science 
This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.
This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Characterization of Nonribosomal Peptide Synthetases with NRPSsp
Bioinformatic sequence analysis allows the functional characterization of newly sequenced proteins. Nonribosomal peptide synthetases (NRPSs) are multi-modular enzymes involved in the biosynthesis of natural products. The current omics era has enabled the exponential growth of the sequenced NRPS, and it is important to characterize the final product of these synthetases. Here, how to achieve the prediction of substrates which bind to adenylation domains in NRPS with NRPSsp (
www.nrpssp.com
) bioinformatic tool is described. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
Alignment-Free Methods for the Detection and Specificity Prediction of Adenylation Domains
Identifying adenylation domains (A-domains) and their substrate specificity can aid the detection of nonribosomal peptide synthetases (NRPS) at genome/proteome level and allow inferring the structure of oligopeptides with relevant biological activities. However, that is challenging task due to the high sequence diversity of A-domains (~10–40 % of amino acid identity) and their selectivity for 50 different natural/unnatural amino acids. Altogether these characteristics make their detection and the prediction of their substrate specificity a real challenge when using traditional sequence alignment methods, e.g., BLAST ...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics
Liquid chromatography–mass spectrometry (LC-MS)-based proteomics is a powerful technique for the profiling of protein expression in cells in a high-throughput fashion. Herein we report a protocol using LC-MS/MS-based proteomics for the screening of enzymes involved in natural product biosynthesis, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) from bacterial strains. Taking advantage of the large size of modular NRPSs and PKSs (often>200 kDa), size-based separation (SDS-PAGE) is employed prior to LC-MS/MS analysis. Based upon the protein identifications obtained through software s...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
The Use of ClusterMine36 for the Analysis of Polyketide and Nonribosomal Peptide Biosynthetic Pathways
Polyketides and nonribosomal peptides constitute two large families of microbial natural products. Over the past 20 years a broad range of microbial polyketide and nonribosomal peptide biosynthetic pathways have been characterized leading to a surfeit of genetic data on polyketide and nonribosomal peptide biosynthesis. We developed the ClusterMine360 database, which stores the antiSMASH-based annotation of gene clusters in the NCBI database, linking the structure of the natural product to the biosynthetic gene cluster. This database is searchable and enables the user to access multiple sequence files for phylogenetic analy...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
Bioinformatics Tools for the Discovery of New Nonribosomal Peptides
This chapter helps in the use of bioinformatics tools relevant to the discovery of new nonribosomal peptides (NRPs) produced by microorganisms. The strategy described can be applied to draft or fully assembled genome sequences. It relies on the identification of the synthetase genes and the deciphering of the domain architecture of the nonribosomal peptide synthetases (NRPSs). In the next step, candidate peptides synthesized by these NRPSs are predicted in silico, considering the specificity of incorporated monomers together with their isomery. To assess their novelty, the two-dimensional structure of the peptides can be c...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
Annotating and Interpreting Linear and Cyclic Peptide Tandem Mass Spectra
Nonribosomal peptides often possess pronounced bioactivity, and thus, they are often interesting hit compounds in natural product-based drug discovery programs. Their mass spectrometric characterization is difficult due to the predominant occurrence of non-proteinogenic monomers and, especially in the case of cyclic peptides, the complex fragmentation patterns observed. This makes nonribosomal peptide tandem mass spectra annotation challenging and time-consuming. To meet this challenge, software tools for this task have been developed. In this chapter, the workflow for using the software mMass for the annotation of experim...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
The Continuing Development of E. coli as a Heterologous Host for Complex Natural Product Biosynthesis
Heterologous biosynthesis of natural products is meant to enable access to the vast array of valuable properties associated with these compounds. Often motivated by limitations inherent in native production hosts, the heterologous biosynthetic process begins with a candidate host regarded as technically advanced relative to original producing organisms. Given this requirement, E. coli has been a top choice for heterologous biosynthesis attempts as associated recombinant tools emerged and continue to develop. However, success requires overcoming challenges associated with natural product formation, including complex biosynt...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
Reconstitution of Fungal Nonribosomal Peptide Synthetases in Yeast and In Vitro
The emergence of next-generation sequencing has provided new opportunities in the discovery of new nonribosomal peptides (NRPs) and NRP synthethases (NRPSs). However, there remain challenges for the characterization of these megasynthases. While genetic methods in native hosts are critical in elucidation of the function of fungal NRPS, in vitro assays of intact heterologously expressed proteins provide deeper mechanistic insights in NRPS enzymology. Our previous work in the study of NRPS takes advantage of Saccharomyces cerevisiae strain BJ5464-npgA as a robust and versatile platform for characterization of fungal NRPSs. H...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
Facile Synthetic Access to Glycopeptide Antibiotic Precursor Peptides for the Investigation of Cytochrome P45 Action in Glycopeptide Antibiotic Biosynthesis
The glycopeptide antibiotics are an important class of complex, medically relevant peptide natural products. Given that the production of such compounds all stems from in vivo biosynthesis, understanding the mechanisms of the natural assembly system—consisting of a nonribosomal-peptide synthetase machinery (NRPS) and further modifying enzymes—is vital. In order to address the later steps of peptide biosynthesis, which are catalyzed by Cytochrome P450s that interact with the peptide-producing nonribosomal peptide synthetase, peptide substrates are required: these peptides must also be in a form that can be conju...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
Colorimetric Detection of the Adenylation Activity in Nonribosomal Peptide Synthetases
Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes consisting of catalytic domains. The substrate specificities of adenylation (A) domains determine the amino-acid building blocks to be incorporated during nonribosomal peptide biosynthesis. The A-domains mediate ATP-dependent activation of amino-acid substrates as aminoacyl-O-AMP with pyrophosphate (PPi) release. Traditionally, the enzymatic activity of the A-domains has been measured by radioactive ATP–[32P]-PPi exchange assays with the detection of 32P-labeled ATP. Recently, we developed a colorimetric assay for the direct detection of PPi as a ye...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains
A series of inhibitors have been designed based on 5′-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of l-Phe-AMS (l-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert l-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
Secondary Metabolic Pathway-Targeted Metabolomics
This chapter provides step-by-step methods for building secondary metabolic pathway-targeted molecular networks to assess microbial natural product biosynthesis at a systems level and to aid in downstream natural product discovery efforts. Methods described include high-resolution mass spectrometry (HRMS)-based comparative metabolomics, pathway-targeted tandem MS (MS/MS) molecular networking, and isotopic labeling for the elucidation of natural products encoded by orphan biosynthetic pathways. The metabolomics network workflow covers the following six points: (1) method development, (2) bacterial culture growth and organic...
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay
We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - May 8, 2016 Category: Biochemistry Source Type: news
In Situ Analysis of Bacterial Lipopeptide Antibiotics by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a technique developed in the late 1990s enabling the two-dimensional mapping of a broad variety of biomolecules present at the surface of a sample. In many applications including pharmaceutical studies or biomarker discovery, the distribution of proteins, lipids or drugs, and metabolites may be visualized within tissue sections. More recently, MALDI MSI has become increasingly applied in microbiology where the versatility of the technique is perfectly suited to monitor the metabolic dynamics of bacterial colonies. The work described here i...
Source: Springer protocols feed by Protein Science - February 6, 2016 Category: Biochemistry Source Type: news
The Assembly Line Enzymology of Polyketide Biosynthesis
Polyketides are a structurally and functionally diverse family of bioactive natural products that have found widespread application as pharmaceuticals, agrochemicals, and veterinary medicines. In bacteria complex polyketides are biosynthesized by giant multifunctional megaenzymes, termed modular polyketide synthases (PKSs), which construct their products in a highly coordinated assembly line-like fashion from a pool of simple precursor substrates. Not only is the multifaceted enzymology of PKSs a fascinating target for study, but it also presents considerable opportunities for the reengineering of these systems affording a...
Source: Springer protocols feed by Protein Science - February 6, 2016 Category: Biochemistry Source Type: news
