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Halogenation of unactivated carbon centers in natural product biosynthesis: Trichlorination of leucine during barbamide biosynthesis

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Journal of the American Chemical Society (Impact Factor: 11.44). 04/2006; 128(12):3900-1. DOI: 10.1021/ja060151n
Source: PubMed

ABSTRACT The in vitro reconstitution of leucine halogenation during barbamide biosynthesis has been accomplished. It has been demonstrated that the triple chlorination of the unactivated pro-R methyl group of the peptidyl carrier protein-tethered l-Leu substrate is carried out by the tandem action of two nonheme iron(II)-dependent halogenases, BarB1 and BarB2. Investigation of the substrate specificities of each of the halogenating enzymes revealed their complementary roles in the generation of trichloroleucine.

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    ABSTRACT: Naturstoffe mit einem außerordentlich breiten Spektrum an Strukturen und pharmakologischen Bedeutungen werden fließbandartig an multimodular aufgebauten nichtribosomalen Peptidsynthetasen (NRPS) assembliert. Jedes Modul dieser Megaenzyme umfasst dabei eine Anzahl von katalytisch eigenständigen Domänen, deren spezifische Interaktion den Einbau und die Modifikation eines Bausteins sichert. Sehr häufig enthalten die Produkte D-Aminosäuren, die zum Großteil durch modulintegrierte Epimerisierungs- (E-) Domänen aus den entsprechenden über einen Thioester peptidyl carrier protein (PCP) gebundenen L-Intermediaten in einer Gleichgewichtsreaktion erzeugt werden. Im Rahmen dieser Arbeit wurde ein chemoenzymatischer Ansatz zur quantitativen Untersuchung der nativen Substratspezifität von vier unterschiedlichen Synthetasen entstam-menden E-Domänen entwickelt. Dazu wurden entsprechende PCP-E-Didomänen auf geneti-scher Ebene isoliert. Zwei dieser Konstrukte (TycB3- und FenD2-PCP-E) enstammten Elongationsmodulen und enthielten sogenannte Peptidyl-E-Domänen, während die beiden anderen (TycA- und GrsA-PCP-E) Initiationsmodulen entnommen wurden und entsprechend Aminoacyl-E-Domänen trugen. Die Apo-Proteine wurden überproduziert und unter Verwen-dung der promiskuitiven 4´-Phophopantethein- (Ppant-) Transferase Sfp mit einer Auswahl synthetisierter Peptidyl-CoAs fehlmodifiziert. Enzymgebundene Peptidyl-S-Ppant Reaktions-produkte der darauffolgenden, durch die E-Domänen katalysierten Stereoinversion wurden chemisch abgespalten und auf ihr L zu D-Verhältnis untersucht. Wie anhand von jeweils berechneten kobs-Werten und vorgefundenen L/D-Endgleichgewichten evaluiert wurde, tolerieren alle vier getesteten E-Domänen eine Vielfalt von Peptidyl-S-Ppant-Substraten. Es zeigte sich, dass kein Zusammenhang zwischen der Spezifität (aktivierter Aminosäure) eines Moduls und der Toleranz der ihm entstammenden E-Domäne existiert. Veränderungen am N-terminalen Ende des Substrats können dabei gleiche Auswirkungen auf die Epimerisierungs-aktivität haben wie Variationen des C-Terminus (der Thioester gebundenen Aminosäure). Die E-Domänen invertierten N-methylierte Substrate besonders effizient. Hervorzuheben ist die entdeckte Fähigkeit von Aminoacyl-E-Domänen zur Epimerisierung von Peptidyl-Substraten. Außerdem konnte deutlich gemacht werden, dass die Kondensations (C-) Domäne von TycB1 ebenso in der Lage ist, Peptidyl-Substrate von TycA zu empfangen und zu prozessieren. Da E-Domänen in vielen Systemen am C-Terminus einer Synthetase stehen, wurde angenommen, dass sie in dieser Position an der gezielten Interaktion mit dem nachfolgenden Enzym und dem geregelten Transfer von Intermediaten beteiligt sind. Deshalb setzte sich der zweite Teil der vorliegenden Arbeit mit der Beleuchtung dieser funktionellen Rolle auseinander. Es wurden dimodulare Derivate aus der Tyrocidin-Synthetase B auf genetischer Ebene konstruiert und die überproduzierten rekombinanten Proteine charakterisiert. Zunächst wurde eine optimierte Abfolge aus tryptischer Proteolyse und hochauflösender Massenspek-trometrie eingesetzt, um die Bildung von Intermediaten am TycB3-PCP des TycB2-3-Wildtyps und des E-Domänen-Austauschproteins TycB2-3-AT.CAT/EtycA direkt zu verfolgen. Der E-Domänen-Austausch beeinträchtigte die Dipeptid- (Phe-Phe-) Bildung signifikant. Ferner wurden die beiden genannten Enzyme und ein weiteres, bei dem die Kommunikation vermittelnde (COM-) Domäne gegen die entsprechende von TycA ausgetauscht worden war, mit dem Akzeptor TycB1 getestet. Funktionelle Unterschiede zwischen Peptidyl- und Aminoacyl-E-Domänen im Kontext der in trans Interaktion wurden hierbei deutlich. Peptidyl-E-Domänen scheinen für die Regulation von Kondensation, Epimerisierung und Transfer von Zwischenstufen zum downstream Modul optimiert zu sein, während Aminoacyl-E-Domänen die upstream Kondensation beeinträchtigen und Misinitiation hervorrufen. Diese Erkenntnisse werden für die erfolgreiche Gestaltung neuer Wirkstoffe per Biokombinatorik mit NRPS unter Einbeziehung von E-Domänen von Bedeutung sein. A broad variety of structurally diverse and pharmacologically relevant natural compounds is synthesized by multimodular nonribosomal peptide synthetase (NRPS) assembly lines. Each module within these megaenzyme systems comprises a number of domains that have distinct catalytic functions and interact specifically to ensure the incorporation and modification of one building block. In many cases the products contain D-amino acids, the majority of which is generated in an equilibrium-forming reaction by module integrated epimerization (E) domains from the corresponding peptidyl carrier protein- (PCP-) bound L-intermediates. Within the scope of this work a chemoenzymatic approach was developed in order to investigate the native substrate specificity of four E domains derived from different synthetases. For this purpose, corresponding PCP-E bidomains were isolated at gene level. Two of these constructs (TycB3- and FenD2-PCP-E) originated from elongation modules containing so-called peptidyl-E domains, while the two other (TycA- and GrsA-PCP-E) were derived from initiation modules accordingly including aminoacyl-E domains. The recombinant apo-proteins were overproduced and modified with a selection of peptidyl-CoAs utilizing the promiscuous 4´-phosphopantetheine- (Ppant-) transferase Sfp. Enzyme-bound peptidyl-S-Ppant reaction products of the subsequently occurring stereoinversion catalyzed by the E domains were cleaved chemically and analyzed for their L to D-ratio. Individually calculated kobs values and final L/D-equilibria observed for each reaction showed that all four E domains tolerate various peptidyl-S-Ppants. Apparently, the substrate tolerance of an E domain does not correlate with the specificity of the module (activated amino acid) it originates from. The epimerization efficiency can equally be affected by alterations of the substrate at the C-terminus (directly thioester-bound amino acid) and the N-terminal part. All tested E domains converted N-methylated substrates with great activity. The most interesting observation was the ability of aminoacyl-E domains to epimerize peptidyl-substrates. Additionally, it could be demonstrated that the condensation (C) domain of TycB1 is able to elongate peptidyl-substrates transferred from TycA. In several systems the C-termini of synthetases harbor E domains, which in these cases seem to facilitate the ordered interaction with the following enzyme and the directed transfer of intermediates. Therefore, the second part of the work presented here aimed at elucidating this additional functional role. Bimodular derivatives of the tyrocidine synthetase B were constructed at gene level and the overproduced recombinant proteins were characterized. Initially, sequent tryptic proteolysis and high-resolution mass spectrometry were optimized for the direct interrogation of intermediates attached to the TycB3-PCP domain of the wild-type TycB2-3 and the E domain exchange protein TycB2-3-AT.CAT/EtycA. The E domain exchange significantly affected dipeptide- (Phe-Phe-) formation. In addition, the two mentioned proteins and a version of TycB2-3 fused to the communication-mediating (COM) domain of TycA were applied in product formation assays with the acceptor TycB1 to corroborate E domain impact on intermodular NRPS action. Significant functional differences in terms of in trans interaction and misinitiation were observed between the two types of C-terminal E domains. Peptidyl-E domains seem to be optimized for regulating the progression of peptide bond formation, epimerization, and intermediate transfer to the downstream module, whereas aminoacyl-E domains impair upstream condensation and cause misinitiation. These perceptions will be of fundamental importance for the successful generation of novel bioactive compounds by biocombinatorial rearrangements of NRPSs involving E domains.
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    ABSTRACT: Mass spectrometry(MS) alone, or combined with chemical cross-linking and proteolysis, is a valuable method in protein characterization. However, detection of cross-linked peptides remains challenging due to ion suppression and cross-linked peptide signals are difficult to differentiate from those of unmodified peptides. In this thesis, efforts to design a novel chemical cross-linker with metal oxide enrichment potential or unique gas-phase fragmentation pattern are presented. A series of biological studies, including mapping of protein-peptide interactions in the transcriptional machinery and determination of enzyme function and functional differentiation in natural product biosynthesis, were also performed. Photo-cross-linking combined with multiplexed MS and biotin-avidin enrichment was employed to map binding sites between gene transcriptional activators and a co-activator(mediator) with resolution up to one amino acid residue. Unprecedented C-C and C-S bond cleavages were observed in matrix-assisted laser desorption/ionization tandem time-of-flight collision induced dissociation(MALDI TOF/TOF CID) of photo-cross-linked products. A novel phosphate-containing cross-linker was synthesized and demonstrated to have amine reactivity. The corresponding cross-linked product was selectively enriched by metal dioxides.Gas-phase fragmentation patterns of peptides linked with disulfide-containing cross-linkers were examined in electron capture dissociation(ECD), electron detachment dissociation(EDD), negative ion CID, and MALDI TOF/TOF CID, to find an optimal gas-phase cleavage method for identification of such cross-linked peptides.Iodinated peptides were subjected to ECD and electron transfer dissociation(ETD), showing highly selective C-I bond cleavage.A proximate, either through bond or through space, positive charge appears to facilitate C-I bond cleavage.These results provide a guide to design novel gas-phase cleavable cross-linkers and an additional model for elucidating the ECD/ETD mechanism. Quadrupole fractionation was also applied in this thesis to enhance detection in the 350-600 m/z region. Fourier transform ion cyclotron resonance(FT-ICR) MS and infrared multiphoton dissociation(IRMPD) were applied to characterize the function and functional differentiation of enzymes from natural product biosynthetic pathways. GCN5-related N-acetyltransferase and enoyl reductase(ER) involved in Curacin A(Cur) biosynthesis were shown to have the unprecedented functions of dual decarboxylation/thiol acetyl transfer and cyclopropanation, respectively. Catalytic efficiencies of Cur and Jamaicamide ERs and site-specific mutants of Cur halogenase were measured by IRMPD. The role of a sulfotransferase in decarboxylative chain termination in Cur biosynthesis was also elucidated.
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    ABSTRACT: Barbamide is a mixed polypeptide-polyketide natural product that contains an unusual trichloromethyl group. The origin of the trichloromethyl group was previously shown to be through chlorination of the pro-R methyl group of L-leucine. Trichloroleucine is subsequently decarboxylated and oxidized to trichloroisovaleric acid and then extended with an acetate unit to form the initial seven carbons of barbamide. In this study we used a combination of biosynthetic feeding experiments and enzymatic analysis to characterize the initial steps required for formation of trichloroleucine and its chain-shortened product, trichloroisovaleric acid. Results from isotope-labeled feeding experiments showed that both dichloroleucine and trichloroleucine are readily incorporated into barbamide; however, monochloroleucine is not. This suggests that halogenation of the pro-R methyl group of leucine occurs as two discrete reactions, with the first involving incorporation of at least two halogen atoms and the second converting dichloroleucine to trichloroleucine. Additionally, the initial tandem dichlorination must occur before substrate can be further processed by the remainingbar pathway enzymes. In vitro analysis of the first five open reading frames (ORFs; barA, barB1, barB2, bar C, barD) of the barbamide gene cluster has yielded new insights into the processing of leucine to form the trichloroisovaleryl-derived unit in the final product.
    Journal of Natural Products 07/2006; 69(6):938-44. DOI:10.1021/np050523q · 3.95 Impact Factor
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