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An orthogonal active site identification system (OASIS) for proteomic profiling of natural product biosynthesis.

ABSTRACT: A significant gap exists between genetics-based investigations of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways and our understanding of their regulation, interaction, and activity in living systems. To help bridge this gap, here we present an orthogonal active site identification system (OASIS) for the proteomic identification and analysis of PKS/NRPS biosynthetic enzymes. OASIS probes target conserved features of PKS/NRPS active sites to provide activity-based enrichment of modular synthases, followed by analysis through multidimensional protein identification technology (MudPIT) LC-MS/MS analysis. When applied to the model bacterium Bacillus subtilis, this functional proteomics method detects and quantifies all four modular synthases in the organism. Furthermore, tandem application of multiple OASIS probes enhances identification of specific PKS/NRPS modules from complex proteomic mixtures. By expanding the dynamic range of proteomic analysis for PKS/NRPS enzymes, OASIS offers a valuable tool for strain comparison, culture condition optimization, and enzyme discovery.

SUBMITTER: Meier JL

PROVIDER: S-EPMC2818308 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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An orthogonal active site identification system (OASIS) for proteomic profiling of natural product biosynthesis.

Meier Jordan L JL Niessen Sherry S Hoover Heather S HS Foley Timothy L TL Cravatt Benjamin F BF Burkart Michael D MD

ACS chemical biology 20091101 11

A significant gap exists between genetics-based investigations of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways and our understanding of their regulation, interaction, and activity in living systems. To help bridge this gap, here we present an orthogonal active site identification system (OASIS) for the proteomic identification and analysis of PKS/NRPS biosynthetic enzymes. OASIS probes target conserved features of PKS/NRPS active sites to provide act ...[more]

PMID: 19785476

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Project description:Natural products are molecules that fulfil a range of important ecological functions. Many natural products have been exploited for pharmaceutical and agricultural applications. In contrast to many other specialised metabolites, the products of modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) systems can often (partially) be predicted from the DNA sequence of the biosynthetic gene clusters. This is because the biosynthetic pathways of NRPS and PKS systems adhere to consistent rulesets. These universal biosynthetic rules can be leveraged to generate biosynthetic models of biosynthetic pathways. While these principles have been largely deciphered, software that leverages these rules to automatically generate visualisations of biosynthetic models has not yet been developed. To enable high-quality automated visualisations of natural product biosynthetic pathways, we developed RAIChU (Reaction Analysis through Illustrating Chemical Units), which produces depictions of biosynthetic transformations of PKS, NRPS, and hybrid PKS/NRPS systems from predicted or experimentally verified module architectures and domain substrate specificities. RAIChU also boasts a library of functions to perform and visualise reactions and pathways whose specifics (e.g., regioselectivity, stereoselectivity) are still difficult to predict, including terpenes, ribosomally synthesised and posttranslationally modified peptides and alkaloids. Additionally, RAIChU includes 34 prevalent tailoring reactions to enable the visualisation of biosynthetic pathways of fully maturated natural products. RAIChU can be integrated into Python pipelines, allowing users to upload and edit results from antiSMASH, a widely used BGC detection and annotation tool, or to build biosynthetic PKS/NRPS systems from scratch. RAIChU's cluster drawing correctness (100%) and drawing readability (97.66%) were validated on 5000 randomly generated PKS/NRPS systems, and on the MIBiG database. The automated visualisation of these pathways accelerates the generation of biosynthetic models, facilitates the analysis of large (meta-) genomic datasets and reduces human error. RAIChU is available at https://github.com/BTheDragonMaster/RAIChU and https://pypi.org/project/raichu .Scientific contributionRAIChU is the first software package capable of automating high-quality visualisations of natural product biosynthetic pathways. By leveraging universal biosynthetic rules, RAIChU enables the depiction of complex biosynthetic transformations for PKS, NRPS, ribosomally synthesised and posttranslationally modified peptide (RiPP), terpene and alkaloid systems, enhancing predictive and analytical capabilities. This innovation not only streamlines the creation of biosynthetic models, making the analysis of large genomic datasets more efficient and accurate, but also bridges a crucial gap in predicting and visualising the complexities of natural product biosynthesis.

Dataset Information

An orthogonal active site identification system (OASIS) for proteomic profiling of natural product biosynthesis.

Publications

An orthogonal active site identification system (OASIS) for proteomic profiling of natural product biosynthesis.

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