The mycosubtilin synthetase of
            <i>Bacillus subtilis</i>
            ATCC6633: A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase

Duitman, Erwin H.; Hamoen, Leendert W.; Rembold, Martina; Venema, Gerard; Seitz, Harald; Saenger, Wolfram; Bernhard, Frank; Reinhardt, Richard; Schmidt, Manuel; Ullrich, Christian; Stein, Torsten; Leenders, Frank; Vater, Joachim

doi:10.1073/pnas.96.23.13294

Cited by 257 publications

(266 citation statements)

References 40 publications

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“…These results also show that this approach can be used for the direct visualization of species that have distinctive isotopic signatures without the need for protein expression in 13 C, 15 N double-depleted media (9). As example, the chloropyrrolyl-S-PltL as well dichloropyrrolyl-S-PltL phosphopantetheinyl ejection ion show the A+2 isotope that comes from 37 chlorine (figures 1D and E) which comprises 24% of the chlorine in nature.…”

Section: Resultsmentioning

confidence: 86%

“…We provide support for the robustness and generality of this assay via 33 examples as this approach was used to characterize the loading of substrates, intermediates and products of proteins involved in the biosynthesis of the antibiotic nikkomycin (8), the antifungal agent pyoluteorin (9,10), the antibiotic prodigiosin (11,12), the antibacterial gramicidin (13,14), the antibacterial agent mycosubtilin (15,16), and the siderophore enterobactin (17,18). Further, intermediates were detected via this gas phase elimination on four proteins from the orphan orphan pksX cluster of B. subtilis that produces an unknown antibiotic (19)(20)(21).…”

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confidence: 99%

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

et al. 2006

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With the emergence of drug resistance and the genomic revolution there has been a renewed interest in the genes that are responsible for the generation of bioactive natural products. Secondary metabolites of one major class are biosynthesized at one or more sites by ultra large enzymes that carry covalent intermediates on phosphopantetheine arms. Because such intermediates are difficult to characterize in vitro, we have developed a new approach for streamlined detection of substrates, intermediates and products attached to a phosphopantetheinyl arm of the carrier site. During vibrational activation of gas phase carrier domains, facile elimination occurs in benchtop and FourierTransform mass spectrometers alike. Phosphopantetheinyl ejections quickly reduce >100 kDa megaenzymes to <1000 Da ions for structural assignment of intermediates at <0.007 Da mass accuracy without proteolytic digestion. This "Top Down" approach quickly illuminated diverse acylintermediates on the carrier domains of the nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs) found in the biosynthetic pathways of prodigiosin, pyoluteorin, mycosubtilin, nikkomycin, enterobactin, gramicidin and several proteins from the orphan pksX gene cluster from Bacillus subtilis. By focusing on just those regions undergoing covalent chemistry, the method delivered clean proof for the reversible dehydration of hydroxymethylglutaryl-S-PksL via incorporation of 2 H or 18 O from the buffer. The facile nature of this revised assay will allow diverse laboratories to spearhead their NRPS/PKS projects with benchtop mass spectrometers.About 50% of today's drugs and 75% of today's antimicrobials are derived from secondary metabolites (1,2). Many of those secondary metabolites are of polyketide or nonribosomal peptide origin. With the emergence of resistance and the genomic revolution there is a "renaissance" ongoing in the discovery of bioactive natural products and the characterization of the genes responsible for their production (1). Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are large enzymes often >>100 kDa that biosynthesize their natural products (e.g., the antibiotics penicillin, vancomycin) via covalent intermediates on phosphopantetheine arms (3). Currently, even when NRPS and PKS proteins can be overproduced their direct interrogation by mass spectrometry (MS) is difficult and timeconsuming, with reports from relatively few laboratories appearing in the primary literature (4-6). Therefore it would be of great benefit for the NRPS and PKS community if new MSbased methods to characterize these proteins were developed and easier to implement. This paper describes such a method.The purpose of this paper is four-fold: 1) the paper introduces a new and efficient method that utilizes a gas phase elimination reaction that takes place during tandem mass spectrometry (MS/MS) to quickly characterize substrates, intermediates and products that are loaded onto the phosphopantetheinyl arm on carrier domains of NRPS...

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Section: Resultsmentioning

confidence: 86%

mentioning

confidence: 99%

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

et al. 2006

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“…Gene prediction algorithms uncovered 4062 genes encoding proteins and 107 encoding structural RNAs. Several previously characterized features were recognized in the sequence, including defective prophage PBSZ (Seaman et al, 1964), the tryptophan operon region (Zeigler et al, 2008), the ribitol teichoic acid gene cluster (Lazarevic et al, 2002) and the biosynthetic gene clusters for the peptide antibiotics rhizocticin (Borisova et al, 2010) and mycosubtilin (Duitman et al, 1999).…”

Section: Resultsmentioning

confidence: 99%

The genome sequence of Bacillus subtilis subsp. spizizenii W23: insights into speciation within the B. subtilis complex and into the history of B. subtilis genetics

Zeigler

2011

Microbiology

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The genome sequence of Bacillus subtilis subsp. spizizenii W23: insights into speciation within the B. subtilis complex and into the history of B. subtilis genetics The genome sequence of Bacillus subtilis subsp. spizizenii W23 has been determined. The sequence strongly suggests that W23 is a direct descendant of B. subtilis ATCC 6633. W23 shares a 3.6 Mb core genome with the intensively studied model organism B. subtilis subsp. subtilis 168, and gene order within this core has been strongly conserved. Additionally, the W23 genome has 157 accessory (that is, non-core) genome segments that are not found in 168, while the 168 genome has 141 segments not found in W23. The distribution of sequences similar to these accessory segments among other genomes of the B. subtilis species complex shows that those sequences having entered into the phylogeny of the complex more recently tend to be larger and more AT-rich than those having entered earlier. A simple model can account for these observations, in which parasitic or symbiotic DNAs are transferred into the genome and then are reduced in size and modified in base composition during speciation. INTRODUCTIONStrain W23 holds an important place in the history of Bacillus subtilis genetics. The development of B. subtilis as a bacterial model system began over 60 years ago with the pioneering mutagenesis studies of Burkholder & Giles (1947). Strain 168, a tryptophan auxotrophic mutant isolated in those experiments, was subsequently found to be naturally competent for genetic transformation (Spizizen, 1958). This discovery quickly spawned a research community focused on exploring the genetics and development of this Gram-positive sporeformer and on exploiting its biotechnological potential.In addition to strain 168, a handful of other Burkholder and Giles mutants were also preserved (Spizizen, 1984). One of them, strain 23, was a threonine auxotroph that can readily revert to prototrophy (D. R. Zeigler, unpublished results). Strain W23, believed to be one such revertant, was widely viewed as a 'wild-type' counterpart to strain 168. For several years, W23 served as a 'universal donor' and 168 derivatives as 'universal recipients' for strain construction and genetic mapping (Anagnostopoulos & Spizizen, 1961;Nester & Lederberg, 1961). Evidence gradually accumulated, however, that W23 and 168 were fundamentally different in cell wall composition, resident prophages and genome content (Zeigler et al., 2008). It is now clear that 168 is closely related to -and probably a descendant of -the B. subtilis Marburg strain, which is stocked in the American Type Culture Collection (ATCC) as 6051 T and in the National Collection of Industrial, Marine and Food Bacteria (NCIMB) collection as 3610 T (Srivatsan et al., 2008; Zeigler et al., 2008). It is equally clear that W23 must have arisen from an independent isolate, although its exact origin has remained a mystery (Hemphill & Whiteley, 1975).Strain W23 continues to have relevance for the B. subtilis research community. For example, its c...

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“…Although AT-less PKS is very rare and the recently reported putative pederin cluster was the only other example to our knowledge that lacks the cognate AT domain from all modules of a type I PKS (30), individual modules that lack the AT domain have been noted in a few other type I PKS or hybrid NRPS-PKS systems (31)(32)(33)(34)(35).…”

Section: The Interaction Among Lnmg Lnmi and Lnmj Represents A Prevmentioning

confidence: 99%

Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis

Tang

Shen

2003

Proc. Natl. Acad. Sci. U.S.A.

246

259

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Type I polyketide synthases (PKSs) are multifunctional enzymes that are organized into modules, each of which minimally contains a ␤-ketoacyl synthase, an acyltransferase (AT), and an acyl carrier protein. Here we report that the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 consists of two PKS genes, lnmI and lnmJ, that encode six PKS modules, none of which contain the cognate AT domain. The only AT activity identified within the lnm gene cluster is a discrete AT protein encoded by lnmG. Inactivation of lnmG, lnmI, or lnmJ in vivo abolished LNM biosynthesis. Biochemical characterization of LnmG in vitro showed that it efficiently and specifically loaded malonyl CoA to all six PKS modules. These findings unveiled a previously unknown PKS architecture that is characterized by a discrete, iteratively acting AT protein that loads the extender units in trans to ''AT-less'' multifunctional type I PKS proteins for polyketide biosynthesis. This PKS structure provides opportunities for PKS engineering as exemplified by overexpressing lnmG to improve LNM production.

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The mycosubtilin synthetase of Bacillus subtilis ATCC6633: A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase

Cited by 257 publications

References 40 publications

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

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

The genome sequence of Bacillus subtilis subsp. spizizenii W23: insights into speciation within the B. subtilis complex and into the history of B. subtilis genetics

Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis

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