Dissecting non-ribosomal and polyketide biosynthetic machineries using electrospray ionization Fourier-Transform mass spectrometry

Abstract

Many virulence factors and bioactive compounds with antifungal, antimicrobial, and antitumor properties are produced via the non-ribosomal peptide synthetase (NRPS) or polyketide synthase(PKS) paradigm. During the biosynthesis of these natural products, substrates, intermediates and side products are covalently tethered to the NRPS or PKS catalyst, introducing mass changes, making these biosynthetic systems ideal candidates for interrogation by large molecule mass spectrometry. This review serves as an introduction into the application of electrospray ionization Fourier-Transform massspectrometry (ESI-FTMS) to investigate NRPS and PKS systems. ESI-FTMS can be used to understand substrate tolerance, timing of covalent linkages, timing of tailoring reactions and the transfer of substrates and biosynthetic intermediates from domain to domain. Therefore we not only highlight key mechanistic insights for thiotemplate systems as found on the enterobactin,yersiniabactin, epothilone, clorobiocin, coumermycin, pyoluteorin, gramicidin, mycosubtilin, C-1027,6-deoxyerythronolide B and FK520 biosynthetic pathways, but we also explain the approaches taken to identify active sites from complex digests and compare the FTMS based assay to traditional assays and other mass spectrometric techniques. Although mass spectrometry was introduced over two decades ago to investigate NRPS and PKS biosynthetic systems, this is the first review devoted to this methodology.

Biosynthesis of polybrominated aromatic organic compounds by marine bacteria

“…Upon transfer of pyrrolyl- S -CoA ( 16 ) to apo- S -Bmp1(ACP) by the B. subtilis phosphopantetheinyl transferase Sfp (described in the Online Methods section), pyrrolyl- S -Bmp1 is generated, that is mono-, di- and tribrominated by Bmp2 in the presence of NADPH, KBr and E. coli flavin-reductase SsuE. Calculation for MS1 peptide masses and peptide identification protocol, as inspired by prior reports 24,50 is described in detail in the Supplementary Note 1. Note that the expression of Bmp1 in E. coli generates a mixture of apo-Bmp1 and holo- S -Bmp1.…”

Interrogation of Global Active Site Occupancy of a Fungal Iterative Polyketide Synthase Reveals Strategies for Maintaining Biosynthetic Fidelity

“…1,2 On the other hand, iterative PKSs, such as the discrete bacterial enzymes and fungal multidomain systems, use substrate loading and condensation domains in successive catalytic cycles to achieve full-length products. Although iterative and modular PKSs share the same fundamental chain elongation step—the sequential condensation of malonyl derivatives—they have different demands for the coordination of oligoketide assembly.…”

Proteasomal Protein Degradation in Mycobacteria Is Dependent upon a Prokaryotic Ubiquitin-like Protein

“…For the intact protein analysis only LTQ data were used because the protein was poorly resolved by ion cyclotron resonance-mass spectrometry due to the low transmission efficiency of the ions, even at 8000-ms accumulation times, and the preprocessing of the data by the Thermo LTQ-FT-ICR instrument, the intact protein was analyzed with the LTQ portion of the instrument. The data were manually processed by deconvolution as described (15). The error bars reported are the standard deviation observed from the average of all the charge states and fall within the expected mass accuracy of an LTQ instrument when used for intact protein analysis.…”