Activity Screening of Carrier Domains within Nonribosomal Peptide Synthetases Using Complex Substrate Mixtures and Large Molecule Mass Spectrometry

Abstract

For screening a pool of potential substrates that load carrier domains found in non-ribosomal peptide synthetases, large molecule mass spectrometry is shown to be an ideal, unbiased assay. Combining the high resolving power of Fourier-Transform Mass Spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A 1 , yersiniabactin, pyochelin and enterobactin biosynthetic pathways as proof-of-principle, preferred substrates are readily identified from substrate pools. Furthermore this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, this assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown. This is demonstrated by identifying the substrates for two NRPS modules from the genes pksN and pksJ, that are found on an orphan NRPS/PKS hybrid cluster from Bacillus subtilis. This new assay format is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate. KeywordsElectrospray Fourier transform mass spectrometry; Non-ribosomal peptide synthetase; Polyketide synthase; Carrier domains; Post-translational modification Almost weekly, new non-ribosomal peptide synthetase (NRPS) gene clusters encoding for proteins that biosynthesize bioactive compounds are discovered (1-4). Since many of the compounds produced by non-ribosomal peptide synthetase (NRPS) as well as polyketide * To whom correspondence should be addressed, kelleher@scs.uiuc.edu, fax number 217 244-8068 and phone number 217 NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 October 9. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript synthase (PKS) paradigm have potent medicinal utility or are involved in virulence, and display unusual chemistry, they are of great academic and industrial interest. Therefore we wanted to develop an alternative method for substrate screening to complement the more traditional radioactive assays (5,6). In NRPS and PKS systems, the substrates and intermediates are loaded onto and processed while attached to the pantetheinyl functionality on a carrier domain (7,8).Examples of NRPS or hybrid NRPS/PKS natural products for which substrates load onto carrier domains are: the siderophore pyoverdine (9), a virulence factor excreted by pseudomonads, the antimicrobial agents penicillin (10), vancomycin (11) gramicidin (12) and the antitumor agent calicheamycin (13). With ∼300 genomes sequenced and available in the public ...

Convergence of isoprene and polyketide biosynthetic machinery: Isoprenyl- S -carrier proteins in the pksX pathway of Bacillus subtilis

“…Consistent with this proposed series of events, when we treated [2-14 C]Mal-S-AcpK and Acac-S-PksL-T2 with PksF, PksG, PksH, and PksI, we observed stable radiolabeling of PksL-T2. In contrast, when the same experiment was performed with [1,[3][4][5][6][7][8][9][10][11][12][13][14] C]Mal-SAcpK, we observed significant loss of radiolabel from PksL-T2 when PksH and PksI were added ( Fig. 5 and Fig.…”

“…No decarboxylation was detected when Mal-S-PksL-T2 was incubated with PksF, demonstrating that it is selective for Mal-S-AcpK (data not shown). Treatment of [1,[3][4][5][6][7][8][9][10][11][12][13][14] C]Mal-S-AcpK with PksF gave similar results, with rapid PksF-dependent loss of radiolabel observed, to form [1-14 C]Ac-S-AcpK at a rate of 5.5 M⅐min Ϫ1 (Figs. 8 and 9, which are published as supporting information on the PNAS web site).…”

“…We also identify roles for the freestanding carrier protein AcpK and the tandem T-T didomain in PksL. Collections of proteins similar to AcpK, PksFGHI, and tandem carrier domains such as those found in PksL have also been found in the curacin, jamaicamide, mupriocin, antibiotic TA, and pederin biosynthetic clusters (10)(11)(12)(13)(14)(15)(16)(17)(18)(19), and they appear to encode the biosyntheses of natural products that universally contain a single-carbon ␤-branch (18).…”

“…Although the pksX cluster has been proposed to encode the biosynthesis of difficidin (7,9,10), recent work has demonstrated that the NRPS portion of the gene cluster is active, disqualifying difficidin, which does not contain any amines, as a candidate for the biosynthetic product of this cluster (11). Long thought to be cryptic, there is now evidence that it is responsible for the biosynthesis of a product that kills streptomycetes (P. D. Straight, M. A. Fischbach, C.T.W., and R. Kolter, unpublished results).…”

The identification of bacillaene, the product of the PksX megacomplex in Bacillus subtilis

“…The heteronuclear multiple-bond correlation between the C-9 methyl protons and carbonyl C-1Ј link the pentaene/hexaene fragment and triene fragment, and the correlation between the C-16Ј methylene protons and the carbonyl C-1Љ link the pentaene/hexaene and ␣-HIC fragments. Multiple 1 H-13 C heteronuclear multiple-bond correlations between the two amide N-H protons and nearby carbons were observed, and multiple 1 H- 15 N heteronuclear multiple-bond correlations between the two amide nitrogens and nearby protons were observed (SI Figs. 14 and 15).…”