Chemoenzymatic Approaches for Streamlined Detection of Active Site Modifications on Thiotemplate Assembly Lines Using Mass Spectrometry

Abstract

For the direct interrogation of peptides harboring covalently modified serines in nonribosomal peptide synthetases, streamlined methodologies described here employ proteolysis and reporter-coenzyme A analogues of four types. The chromophoric and fluorescent coenzyme A analogues pyrene-maleimidyl-S-CoA and BODIPY-FL-N-(2-aminoethyl)maleimidyl-S-CoA were enzymatically loaded onto the active site serines harbored in the ArCP, PCP1, and PCP2 thiolation domains of PchE and PchF, the nonribosomal peptide synthetases responsible for the biosynthesis of the siderophore pyochelin. During the chromatographic separation of cyanogen bromide digests, observation of the absorbance (at 338 and 504 nm) or fluorescence (after irradiation at 365 nm) enabled the selective detection of peptides containing each active site serine. This resulted in quick detection of each active site peptide by Fourier transform mass spectrometry in the fully reconstituted pyochelin system. The loading of short acyl chain reporters in equimolar quantities permitted further insights into digestion heterogeneity and side reactions by virtue of a mass shift signature on each active site peptide. The chromatographic shift of the reporter-loaded peptides relative to peptides carrying on pathway intermediates was 2 min at 7 kDa, providing a general strategy for efficient localization of "carrier" peptides in complex digests of thiotemplate enzymes. Also, the use of the affinity reporter, biotin-maleimidyl-S-coenzyme A, permitted the isolation of intact synthetases at high purity via removal of contaminating Escherichia coli proteins.

Facile Detection of Acyl and Peptidyl Intermediates on Thiotemplate Carrier Domains via Phosphopantetheinyl Elimination Reactions during Tandem Mass Spectrometry

“…The gradients used were water to acetonitrile gradients as described (9,12,16,18,19,23). During the HPLC runs, the fractions containing the carrier domains were collected, frozen at −80 °C and lyophilized.…”

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

“…CNBr digestion is very useful in the study of NRPS and PKS proteins as it can be done under acidic conditions that are known to significantly slow down the hydrolysis of thioesters. 40,48,49 CNBr digestion generates fewer and larger peptide fragments than most proteases, making the resulting peptide maps less complex. CNBr digestion is usually done with proteins that have been denatured and it should be done in the dark as we have observed increased amounts of oxidations and other unidentified side reactions when exposed to light (personal observations).…”

“…The mass shift reporter relies on loading CoAs that have different masses. 49 These differences can be introduced by the incorporation of stable isotopes or they can be introduced by the incorporation of different substitutents on CoA. As an example, a carrier domain is loaded with a mixture of CoA and This journal is © The Royal Society of Chemistry 2006 acetoacetate-S-CoA at which point the protein is digested, and separated by HPLC.…”

The chemical biology of modular biosynthetic enzymes

“…In order to streamline interrogation of polyketide and nonribosomal peptide biosynthesis by ESI-FTMS, the groups of Walsh and Kelleher developed a method for facile detection of carrier protein active site peptides. 116 Burkart and co-workers observed in 2004 that the substrate tolerance of Sfp allowed transfer of fluorescent CoA analogues onto a variety of carrier proteins. 117 Using this approach, McLoughlin et al loaded modules of the recombinant pyochelin NRPS with fluorescent or UV-detectable CoA analogues.…”

“…117 This provided an effective way of monitoring posttranslational modification of recombinant carrier proteins during coexpression with Sfp. Since this initial report, the labeling of apo-carrier proteins with reporter-labeled CoA analogues has been used to facilitate detection of carrier protein active sites for FTMS based approaches, 116 as well as in a variety of site-specific protein labeling applications, 77,200,201 the subject of a recent review. 202 Perhaps more interestingly in the context of the current discussion, this method showed modest labeling of the native DEBS PKS from proteomic preparations of Saccharopolyspora erythraea, although this result was noted to be dependent on growth conditions.…”