Biosynthesis of the 3,4-dihydroxybenzoate moieties of petrobactin by Bacillus anthracis

The biosynthesis of the 3,4-dihydroxybenzoate moieties of the siderophore petrobactin, produced by B. anthracis str. Sterne, was probed by isotopic feeding experiments in iron-deficient media with a mixture of unlabeled and D-[(13)C6]glucose at a ratio of 5:1 (w/w). After isolation of the labeled siderophore, analysis of the isotopomers was conducted via one-dimensional (1)H and (13)C NMR spectroscopy, as well as (13)C-(13)C DQFCOSY spectroscopy. Isotopic enrichment and (13)C-(13)C coupling constants in the aromatic ring of the isolated siderophore suggested the predominant route for the construction of the carbon backbone of 3,4-DHB (1) involved phosphoenol pyruvate and erythrose-4-phosphate as ultimate precursors. This observation is consistent with that expected if the shikimate pathway is involved in the biosynthesis of these moieties. Enrichment attributable to phosphoenol pyruvate precursors was observed at C1 and C6 of the aromatic ring, as well as into the carboxylate group, while scrambling of the label into C2 was not. This pattern suggests 1 was biosynthesized from early intermediates of the shikimate pathway and not through later shikimate intermediates or aromatic amino acid precursors.     

One metabolite, two pathways: convergence of polypropionate biosynthesis in fungi and marine molluscs

Structural similarity or even the identity of polyketide compounds does not necessarily imply unique biosynthesis. Feeding experiments with a (13)C labeled precursor establish that the C(3) units in 7-methyl-cyercene-1 (1) are derived from intact propionate in the marine mollusc Ercolania funerea. The same compound in the terrestrial fungus Leptosphaeria maculans/Phoma lingam is synthesized by an acetate/SAM pathway thus proving for the first time metabolic convergence of polyketide biosynthesis in eukaryotes. Traditional (1)H-(13)C NMR correlation spectroscopy has been successfully applied to estimate (13)C incorporation in biosynthetic experiments.     

Further Studies on the Biosynthesis of Tabtoxin (Wildfire Toxin): Incorporation of [2,3-13C2]Pyruvate into the β-Lactam Moiety

[2,3-¹³C₂]Pyruvic acid ( 7 ) was synthesized and administered to cultures of Pseudomonas syringae pv tabaci. C(2) and C(3) of 7 were incorporated as an intact unit into the β-lactam moiety of tabtoxin ( 1 ). The result suggests that the biosynthesis of 1 is proceeding in part along the lysine pathway. The labelling pattern in 1 and an incorporation experiment with α,α′-dideuterated (±)-2,6-diaminopimelic acid ( 19 ) indicate that the branching in the biosynthesis of 1 occurs before lysine is formed.     

Characterization of the lnmKLM genes unveiling key intermediates for β-alkylation in leinamycin biosynthesis

Leinamycin (LNM, 1) biosynthesis is proposed to involve β-alkylation of the polyketide intermediate, catalyzed by LnmKLM. Inactivation of lnmK, lnmL, or lnmM afforded mutant strains that accumulated LNM K-1 (2), K-2 (3), K-3 (4), and isomers LNM K-1' (5), K-2' (6), and K-3' (7) whose polyketide origin was established by feeding experiments with sodium [1-(13)C]acetate. These findings confirm the indispensability of LnmKLM in 1 biosynthesis and suggest that β-alkylation proceeds on the growing polyketide intermediate while bound to the LNM polyketide synthase.     

Biosynthesis of tetrapetalones

The biosynthesis of tetrapetalones (tetrapetalones A, B, C, and D) in Streptomyces sp. USF-4727 was studied by feeding experiments with [1-13C] sodium propanoate, [1-13C] sodium butanoate, [carbonyl-13C] 3-amino-5-hydroxybenzoic acid (AHBA) hydrochloride, and [1-13C] glucose, followed by analysis of the 13C-NMR spectra. These feeding experiments revealed that the four tetrapetalones were polyketide compounds constructed from propanoate, butanoate, AHBA, and glucose. The tetrapetalone biosynthetic pathway was also suggested in this study. In this pathway, tetrapetalone A (1) is synthesized by polyketide synthase (PKS) using AHBA as a starter unit, then the side chain of 1 is subjected to acetoxylation to produce tetrapetalone B (2). Additionally, 1 is oxidized and transformed into tetrapetalone C (3). In a similar way, 2 is converted to tetrapetalone D (4). Therefore, the biosynthetic relationship of the four tetrapetalones was indicated.     

Biosynthesis of structurally unique fungal metabolite GKK1032A(2): indication of novel carbocyclic formation mechanism in polyketide biosynthesis

The biosynthesis of the antitumor agent GKK1032A(2) (1) has been investigated by administration of isotopically labeled ((13)C and (2)H) precursors to Penicillium sp. GKK1032. These studies showed that the backbone of 1 is constructed from l-tyrosine and a nonaketide chain flanked with five methyl groups probably by a polyketide synthase and a nonribosomal peptide synthetase hybrid. On the basis of the oxidation level of the starter unit and unusual 13-membered macroether formation between the tyrosine hydroxy group and the polyketide chain, novel cyclization mechanisms on the formation of a tricarbocyclic system and a macroether have been proposed. Involvement of a similar type of cyclization in the biosynthesis of structurally related metabolites is discussed.     

Premalbrancheamide: synthesis, isotopic labeling, biosynthetic incorporation, and detection in cultures of Malbranchea aurantiaca

An advanced metabolite, named premalbrancheamide, involved in the biosynthesis of malbrancheamide (1) and malbrancheamide B (2) has been synthesized in double (13)C-labeled form and was incorporated into the indole alkaloid 2 by Malbranchea aurantiaca. In addition, premalbrancheamide has been detected as a natural metabolite in cultures of M. aurantiaca. The biosynthetic implications of these experiments are discussed.     

两个埃博霉素生物合成中间体修饰化合物的合成

为了使用前体定向生物合成技术研究埃博霉素的生物合成过程并对其进行分子改造, 选定了两个中间体修饰化合物2-甲基噻唑-4-羧酸2-乙酰氨基乙硫基硫酯和(E)-2-甲基-3-(2-甲基噻唑-4-基)丙烯酸2-乙酰氨基乙硫基硫酯进行了化学全合成的研究. 合成方法比已有的合成方法更易操作, 经济性更好. 所有化合物经红外光谱、质谱、核磁共振谱及元素分析确证了结构.     

Pentalenolactone biosynthesis. Molecular cloning and assignment of biochemical function to PtlI, a cytochrome P450 of Streptomyces avermitilis

A gene cluster encoding all of the enzymes for the biosynthesis of the antibiotic pentalenolactone (1) has recently been identified in Streptomyces avermitilis. The biosynthetic gene cluster contains the ptlI (SAV2999) gene which encodes a cytochrome P450 (CYP183A1). PtlI was cloned by PCR and expressed in Escherichia coli as a C-terminal His6-tag protein. Recombinant PtlI bound pentalenene (3) with high affinity (KD = 1.44 +/- 0.14 muM). Incubation of recombinant PtlI with (+/-)-3 in the presence of NADPH, E. coli flavodoxin and flavodoxin reductase, and O2 resulted in conversion to a single enantiomer of pentalen-13-al (7), by stepwise allylic oxidation via pentalen-13-ol (6). The steady-state kinetic parameters for the oxidation of pentalenene (3) to pentalen-13-ol (6) were kcat = 0.503 +/- 0.006 min-1 and Km = 3.33+/-0.62 muM for 3.     

N-methylputrescine oxidation during cocaine biosynthesis: study of prochiral methylene hydrogen discrimination using the remote isotope method

[structure: see text] The stereoselectivity of N-methylputrescine (3) oxidation to pyrrolinium ion 4 in Erythroxylum coca during cocaine (1) biosynthesis was studied. The remote isotope method was used to advantage. Each enantiomer of 4-monodeuterated N-methylputrescine served as a precursor for plant feeding. To facilitate mass-spectrometric analysis of products, a 2H3 13C-methyl group was also incorporated into the 4-deuterio-N-methylputrescines. Oxidative deamination of N-methylputrescine was found to be stereoselective; the pro-S hydrogen atom is removed with 6-10:1 selectivity.     

Studies on the formation and incorporation of streptolidine in the biosynthesis of the peptidyl nucleoside antibiotic streptothricin F

Streptothricin F (STF, 1) is a peptidyl nucleoside antibiotic produced by Streptomyces lavendulae. Studies were conducted to address the formation and timing of incorporation of the arginine-derived base streptolidine (4) during the biosynthesis of 1. [guanidino-(13)C]Streptolidine (10) was prepared by modification of an established method and used in whole-cell incorporation experiments. Analysis of the purified STF by (13)C NMR revealed a 1.9% enrichment of the guanidino carbon, confirming 4 as an advanced precursor to 1 and supporting proposals that 1 is assembled via a convergent biosynthetic pathway. To identify advanced intermediates in the conversion of L-arginine to 4, (2S,3R)-[guanidino-(13)C]capreomycidine (32) was prepared from oxazolidine aldehyde (18) via 1,1-dimethylethyl (4R,1'S)-4-(1',3'-diaminopropyl)-2,2-dimethyl-3-oxazolidinecarboxylate (30). Treatment of 30 with Br(13)CN yielded the corresponding diprotected amino alcohol, which was readily converted to 32. The STF isolated from whole-cell incorporation experiments with 32 showed no significant (13)C enrichment at the guanidino carbon. These results suggest that 32 may be an enzyme-bound intermediate, unable to enter the cell, or is not a precursor to STF.     

Biosynthesis of chaetochromin A, a bis(naphtho-gamma-pyrone), in Chaetomium spp

The biosynthesis of chaetochromin A, a metabolite of Chaetomium gracile, has been studied using [13CH3]methionine, sodium [1-13C]acetate, sodium [1,2-13C2]acetate, sodium [1-13C,2,2,2-2H3]acetate, and sodium [1-13C,1,1-18O2]acetate as precursors. The folding pattern of the polyketide chain in chaetochromin A, biosynthesized from sodium [1,2-13C2]acetate as the precursor, was determined to be the same as that of rubrofusarin by carbon-13 nuclear magnetic resonance (13C-NMR) analysis. By using [13CH3]methionine as a precursor, the source of 2-CH3 was determined. When sodium [1-13C,2,2,2-2H3]acetate was fed, a beta-isotope-shifted peak was observed only for carbon 2. In the 13C-NMR spectra of chaetochromin A and of its hexamethyl ether derived from sodium [1-13C,1,1-18O2]acetate, isotope-shifted peaks were observed for carbons 4, 5, 6, 8 and 10a, but not for carbon 2. These results showed that oxygen 1 originated from the same unit of acetate as carbon 10a.     

Biosynthesis of cylindrospermopsin

Studies on the biosynthesis of cylindrospermopsin (1), a potent hepatotoxin associated with the cyanobacterium Cylindrospermopsis raciborskii, indicate that 1 is an acetogenin with guanidinoacetic acid serving as the starter unit of the polyketide chain. Feeding experiments show that C14 and C15 of 1 are derived from C1 and C2 of glycine, respectively, and C4 through C13 arise from five contiguous acetate units attached head to tail. The methyl carbon on C13 originates from the C(1) pool. The starter unit, established by the incorporation of [guanidino-(13)C,alpha-(15)N]-guanidinoacetic acid into N16 and C17 of 1, does not appear to be formed from glycine by known amidination pathways. The origin of the NH-CO-NH segment in the uracil ring is also unknown.     

Roimatacene: an antibiotic against Gram-negative bacteria isolated from Cystobacter ferrugineus Cb G35 (Myxobacteria)

Roimatacene (1) was isolated from the myxobacterium Cystobacter ferrugineus strain Cb G35 in a bioactivity-guided process, by following the antimicrobial activity against Escherichia coli. Since 1 was extremely sensitive to oxygen, a protective isolation and handling protocol was developed, by utilizing the free radical scavenger 4-ethoxyphenol. The structure of 1 was determined by HRMS, 1D and 2D NMR spectroscopy and chemical derivatization to acetonides and Mosher esters to finally establish the absolute configuration. Methionine and acetate were identified as building blocks in the biosynthesis of 1 by feeding experiments with differently (13)C-labeled precursors. The antimicrobial activity of 1 was determined in a broad screening revealing 1 to inhibit several Gram-negative bacteria.     

Biosynthesis and structural revision of neomarinone

[reaction: see text] The biosynthesis of the meroterpenoid neomarinone from a marine actinomycete was probed through feeding experiments with (13)C-labeled precursors. NMR characterization of [U-(13)C(6)]glucose-enriched neomarinone led to the structural revision of structure 4a to 4b, which was confirmed by extensive 2D NMR spectrometry with unlabeled compound.     

The biosynthesis of the thiazole phosphate moiety of thiamin: the sulfur transfer mediated by the sulfur carrier protein ThiS

Thiamin-pyrophosphate is an essential cofactor in all living systems. The biosynthesis of both the thiazole and the pyrimidine moieties of this cofactor involves new biosynthetic chemistry. Thiazole-phosphate synthase (ThiG) catalyses the formation of the thiazole moiety of thiamin-pyrophosphate from 1-deoxy-D-xylulose-5-phosphate (DXP), dehydroglycine and the sulfur carrier protein (ThiS), modified on its carboxy terminus as a thiocarboxylate (ThiS-thiocarboxylate). Thiazole biosynthesis is initiated by the formation of a ThiG/DXP imine, which then tautomerizes to an amino-ketone. In this paper we study the sulfur transfer from ThiS-thiocarboxylate to this amino-ketone and trap a new thioenolate intermediate. Surprisingly, thiazole formation results in the replacement of the ThiS-thiocarboxylate sulfur with an oxygen from DXP and not from the buffer, as shown by electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) using (18)O labeling of the 13C-, 15N-depleted protein. These observations further clarify the mechanism of the complex thiazole biosynthesis in bacteria.     

Ring A of nukacin ISK-1: a lipid II-binding motif for type-A(II) lantibiotic

Ring A of nukacin ISK-1, which is also present in different type-A(II) lantibiotics, resembles a lipid II-binding motif (TxS/TxD/EC, x denotes undefined residues) similar to that present in mersacidin (type-B lantibiotics), which suggests that nukacin ISK-1 binds to lipid II as a docking molecule. Results from our experiments on peptidoglycan precursor (UDP-MurNAc-pp) accumulation and peptide antagonism assays clearly indicated that nukacin ISK-1 inhibits cell-wall biosynthesis, accumulating lipid II precursor inside the cell, and the peptide activity can be repressed by lipid I and lipid II. Interaction analysis of nukacin ISK-1 and different ring A variants with lipid II revealed that nukacin ISK-1 and nukacin D13E (a more active variant) have a high affinity (K(D) = 0.17 and 0.19 μM, respectively) for lipid II, whereas nukacin D13A (a less active variant) showed a lower affinity, and nukacin C14S (a negative variant lacking the ring A structure) exhibited no interaction. Therefore, on the basis of the structural similarity and positional significance of the amino acids in this region, we concluded that nukacin ISK-1 binds lipid II via its ring A region and may lead to the inhibition of cell-wall biosynthesis.     

Biosynthesis of the validamycins: identification of intermediates in the biosynthesis of validamycin A by Streptomyces hygroscopicus var. limoneus.

To study the biosynthesis of the pseudotrisaccharide antibiotic, validamycin A (1), a number of potential precursors of the antibiotic were synthesized in (2)H-, (3)H-, or (13)C-labeled form and fed to cultures of Streptomyces hygroscopicus var. limoneus. The resulting validamycin A from each of these feeding experiments was isolated, purified and analyzed by liquid scintillation counting, (2)H- or (13)C NMR or selective ion monitoring mass spectrometry (SIM-MS) techniques. The results demonstrate that 2-epi-5-epi-valiolone (9) is specifically incorporated into 1 and labels both cyclitol moieties. This suggests that 9 is the initial cyclization product generated from an open-chain C(7) precursor, D-sedoheptulose 7-phosphate (5), by a DHQ synthase-like cyclization mechanism. A more proximate precursor of 1 is valienone (11), which is also incorporated into both cyclitol moieties. The conversion of 9 into 11 involves first epimerization to 5-epi-valiolone (10), which is efficiently incorporated into 1, followed by dehydration, although a low level of incorporation of 2-epi-valienone (15) is also observed. Reduction of 11 affords validone (12), which is also incorporated specifically into 1, but labels only the reduced cyclitol moiety. The mode of introduction of the nitrogen atom linking the two pseudosaccharide moieties is not clear yet. 7-Tritiated valiolamine (8), valienamine (2), and validamine (3) were all not incorporated into 1, although each of these amines has been isolated from the fermentation, with 3 being most prevalent. Demonstration of in vivo formation of [7-(3)H]validamine ([7-(3)H]-3) from [7-(3)H]-12 suggests that 3 may be a pathway intermediate and that the nonincorporation of [7-(3)H]-3 into 1 is due to a lack of cellular uptake. We thus propose that 3, formed by amination of 12, and 11 condense to form a Schiff base, which is reduced to the pseudodisaccharide unit, validoxylamine A (13). Transfer of a D-glucose unit to the 4'-position of 13 then completes the biosynthesis of 1. Other possibilities for the mechanism of formation of the nitrogen bridge between the two pseudosaccharide units are also discussed.     

Identification and characterization of xiamycin A and oxiamycin gene cluster reveals an oxidative cyclization strategy tailoring indolosesquiterpene biosynthesis

Xiamycin A (XMA) and oxiamycin (OXM) are bacterial indolosesquiterpenes featuring rare pentacyclic ring systems and are isolated from a marine-derived Streptomyces sp. SCSIO 02999. The putative biosynthetic gene cluster for XMA/OXM was identified by a partial genome sequencing approach. Eighteen genes were proposed to be involved in XMA/OXM biosynthesis, including five genes for terpene synthesis via a non-mevalonate pathway, eight genes encoding oxidoreductases, and five genes for regulation and resistance. Targeted disruptions of 13 genes within the xia gene cluster were carried out to probe their encoded functions in XMA/OXM biosynthesis. The disruption of xiaK, encoding an aromatic ring hydroxylase, led to a mutant producing indosespene and a minor amount of XMA. Feeding of indosespene to XMA/OXM nonproducing mutants revealed indosespene as a common precursor for XMA/OXM biosynthesis. Most notably, the flavin dependent oxygenase XiaI was biochemically characterized in vitro to convert indosespene to XMA, revealing an unusual oxidative cyclization strategy tailoring indolosesquiterpene biosynthesis.