The Multiple Carrier Model of Nonribosomal Peptide Biosynthesis at Modular Multienzymatic Templates

Stein, Torsten; Vater, Joachim; Kruft, Volker; Otto, Albrecht; Wittmann‐Liebold, Brigitte; Franke, P.; Panico, Maria; McDowell, Roy A.; Morris, Howard R.

doi:10.1074/jbc.271.26.15428

Cited by 188 publications

(186 citation statements)

References 30 publications

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“…Expression of the corresponding putative P-pant transferase cDNA in E. coli gave rise to a protein that in vitro enhanced low level phosphantetheinylated S2 cell-derived Ebony activity depending on the presence of CoA comparable with the aforementioned Sfp. 2 Our results demonstrate that the higher eucaryote Drosophila has preserved an amino acid activation mechanism that until now was considered to be specific for microbial NRPSs (27). Ebony combines this unique feature with a functional domain that allows peptide bond formation with a structurally constrained group of amines.…”

Section: Discussionmentioning

confidence: 83%

Ebony, a Novel Nonribosomal Peptide Synthetase for β-Alanine Conjugation with Biogenic Amines in Drosophila

Richardt¹,

Kemme²,

Wagner³

et al. 2003

Journal of Biological Chemistry

129

138

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Using Ebony protein either expressed in Escherichia coli or in Schneider S2 cells, we provide evidence for its substrate specificity and reaction mechanism. Ebony activates ␤-alanine to aminoacyladenylate by an adenylation domain and covalently attaches it as a thioester to a thiolation domain in a nonribosomal peptide synthetase (NRPS) related mechanism. In a second reaction, biogenic amines act as external nucleophiles on ␤-alanyl-S-pantetheine-Ebony, thereby releasing in a fast reaction the dipeptide (peptidoamine) in a process that is novel in higher eucaryotes. Therefore, we define Ebony as a ␤-alanyl-biogenic amine synthetase. Insight into the reaction mechanism stems from mutational analysis of an invariant serine that disclosed Ebony as a multienzyme with functional analogy to the starting modules of NRPSs. In light of a putative biogenic amine-deactivating capacity, Ebony function in the nervous system must be reconsidered. We propose that in the Drosophila eye Ebony is involved in the transmission process by inactivation of histamine through ␤-alanyl conjugation.

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Section: Discussionmentioning

confidence: 83%

Ebony, a Novel Nonribosomal Peptide Synthetase for β-Alanine Conjugation with Biogenic Amines in Drosophila

Richardt¹,

Kemme²,

Wagner³

et al. 2003

Journal of Biological Chemistry

129

138

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“…Here we describe a mass spectrometry based method to identify substrates based on mass changes that take place during acylation of a phosphopantetheinyl functionality on the carrier domain(s) of an NRPS protein from very complex substrate reaction mixtures. Observing such acylations by mass spectrometry on carrier domains is now becoming routine (5,19,(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36).The general method to identify covalently loaded substrates or intermediates is shown in Figure 1. Overproduced protein that contains a carrier domain is purified and incubated with Sfp, a promiscuous phosphopantetheinyl transferase from B .subtilis, and CoA in order to generate the holo form of the carrier domain (37).…”

mentioning

confidence: 99%

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

et al. 2006

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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 ...

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“…Analysis of the sequence data has provided insight into the modular construction of their multifunctional polypeptide chains, the occurrence of the modules being associated with the number and the sequence of the amino acid constituents in the peptide product. The reaction sequence involves carboxyl (amino, imino and hydroxy acid) activation in form of an acyladenylate, followed by transfer of the activated acyl moiety to a cysteamine group of an enzyme bound cofactor, 4'-phosphopantetheine (Stein et al, 1996). Sequence infoimation has been obtained for more than 58 peptide synthetase modules, including tyrocidine synthetase 1, gramicidin S synthetase, and &(L-a-aminoadipyl)-L-cysteinyl-D-vahe synthetase (Pavela-Vrancic et al, 1994 a).…”

mentioning

confidence: 99%

The Adenylation Domain of Tyrocidine Synthetase 1

Dieckmann¹,

Pavela-Vrančić²,

Pfeifer³

et al. 1997

European Journal of Biochemistry

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Sequence analysis of peptide synthetases revealed extensive structure similarity with firefly luciferase, whose crystal structure has recently become available, providing evidence for the localization of the active site at the interface between two subdomains separated by a distorted linker region [Conti, E., Franks, N. P. & Brick, P. (1996) Structure 4, 287-2981. The functional importance of two flexible loops, corresponding to the linker region of firefly luciferase and the highly conserved (S/T)GT(T/S)GXPKG core sequence, has been studied in view of the proposed conformational changes by the use of mutant analysis, limited proteolysis and chemical modification of tyrocidine synthetase 1. Substitution of the highly conserved Arg416, residing in the loop separating the subdomains of the adenylation domain, resulted in profound loss of activity. Limited proteolysis of the mutant suggested significant structural changes as manifested by lack of protection to degradation in the presence of substrates, revealing a probable disturbance of the induced-fit mechanism regulating the transformation from an open to a closed conformation. Mutants, obtained by replacement of the conserved Lys186 from the (S/T)GT(T/S)GXPKG core sequence, displayed only minor differences in substrate-binding affinity despite significant reduction of catalytic efficiency. Residue Lys186 appears to play an important role in either stabilization of the bound substrate through charge-charge-interactions, and/or fixing of the loop for maintainance of the active-site conformation.

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The Multiple Carrier Model of Nonribosomal Peptide Biosynthesis at Modular Multienzymatic Templates

Cited by 188 publications

References 30 publications

Ebony, a Novel Nonribosomal Peptide Synthetase for β-Alanine Conjugation with Biogenic Amines in Drosophila

Ebony, a Novel Nonribosomal Peptide Synthetase for β-Alanine Conjugation with Biogenic Amines in Drosophila

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

The Adenylation Domain of Tyrocidine Synthetase 1

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