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Summary Phenazine-type metabolites arise from either phenazine-1-carboxylic acid (PCA) or phenazine-1,6-dicarboxylic acid (PDC). Although the biosynthesis of PCA has been studied extensively, PDC assembly remains unclear. Esmeraldins and saphenamycin, the PDC originated products, are antimicrobial and antitumor metabolites isolated from Streptomyces antibioticus Tu 2706. Herein, the esmeraldin biosynthetic gene cluster was identified on a dispensable giant plasmid. Twenty-four putative esm genes were characterized by bioinformatics, mutagenesis, genetic complementation, and functional protein expressions. Unlike enzymes involved in PCA biosynthesis, EsmA1 and EsmA2 together decisively promoted the PDC yield. The resulting PDC underwent a series of conversions to give 6-acetylphenazine-1-carboxylic acid, saphenic acid, and saphenamycin through a unique one-carbon extension by EsmB1–B5, a keto reduction by EsmC, and an esterification by EsmD1–D3, the atypical polyketide sythases, respectively. Two transcriptional regulators, EsmT1 and EsmT2, are required for esmeraldin production.

The amide synthase of the geldanamycin producer, Streptomyces hygroscopicus, shows a broader chemoselectivity than the corresponding amide synthase present in Actinosynnema pretiosum, the producer of the highly cytotoxic ansamycin antibiotics, the ansamitocins. This was demonstrated when blocked mutants of both strains incapable of biosynthesizing 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase starter unit of both natural products, were supplemented with 3-amino-5-hydroxymethylbenzoic acid instead. Unlike the ansamitocin producer A. pretiosum, S. hygroscopicus processed this modified starter unit not only to the expected 19-membered macrolactams but also to ring enlarged 20-membered macrolactones. The former mutaproducts revealed the sequence of transformations catalyzed by the post-PKS tailoring enzymes in geldanamycin biosynthesis. The unprecedented formation of the macrolactones together with molecular modeling studies shed light on the mode of action of the amide synthase responsible for macrocyclization. Obviously, the 3-hydroxymethyl substituent shows similar reactivity and accessibility toward C-1 of the seco-acid as the arylamino group, while phenolic hydroxyl groups lack this propensity to act as nucleophiles in the macrocyclization. The promiscuity of the amide synthase of S. hygroscopicus was further demonstrated by successful feeding of four other m-hydroxymethylbenzoic acids, leading to formation of the expected 20-membered macrocycles. Good to moderate antiproliferative activities were encountered for three of the five new geldanamycin derivatives, which matched well with a competition assay for Hsp90α.

Ansamitocins are potent antitumor agents produced by Actinosynnema pretiosum. As deduced from their structures, an N-methylation on the amide bond is required among the various modifications. The encoded protein by asm10 belongs to SAM-dependent methyltransferase family. Through gene inactivation and complementation, asm10 was proved to be responsible for the N-methylation of ansamitocins. Asm10 is 33.0 kDa in size and present as monomer as determined by gel filtration. Using N-desmethyl-ansamitocin P-3 as substrate, the optimal temperature and pH were determined to be 32 °C and 10.0 respectively for Asm10 catalysis. Asm10 also showed broad substrate flexibility toward other N-desmethyl ansamycins and synthetic indolin-2-ones. Through site-directed mutagenesis, Asp154 and Leu155 of Asm10 were confirmed to be essential for its catalysis possibly thorough the binding of SAM. The characterization of this unique N-methyltransferase enriched the toolbox for engineering N-methylated derivatives from both natural and synthetic compounds, which will allow modification of known potential drugs.

Asukamycin, a member of the manumycin family metabolites, is an antimicrobial and potential antitumor agent isolated from Streptomyces nodosus subsp. asukaensis. The entire asukamycin biosynthetic gene cluster was cloned, assembled, and expressed heterologously in Streptomyces lividans. Bioinformatic analysis and mutagenesis studies elucidated the biosynthetic pathway at the genetic and biochemical level. Four gene sets, asuA-D, govern the formation and assembly of the asukamycin building blocks: a 3-amino-4-hydroxybenzoic acid core component, a cyclohexane ring, two triene polyketide chains, and a 2-amino-3-hydroxycyclopent-2-enone moiety to form the intermediate protoasukamycin. AsuE1 and AsuE2 catalyze the conversion of protoasukamycin to 4-hydroxyprotoasukamycin, which is epoxidized at C5-C6 by AsuE3 to the final product, asukamycin. Branched acyl CoA starter units, derived from Val, Leu, and Ile, can be incorporated by the actions of the polyketide synthase III (KSIII) AsuC3/C4 as well as the cellular fatty acid synthase FabH to produce the asukamycin congeners A2-A7. In addition, the type II thioesterase AsuC15 limits the cellular level of omega-cyclohexyl fatty acids and likely maintains homeostasis of the cellular membrane.

The conformational properties of a series of pyridoxal amino acid Schiff's bases have been calculated. The calculations were performed using the program CAMSEQ which employs a set of empirical potential energy functions. The cndo/2 charge distributions are also given in this preliminary report. The locations of the local energy minima are consistent with Dunathan's hypothesis that the course of enzymatic reaction is related to the conformation of the pyridoxal phosphate Schiff's base. Calculated results are also in agreement with experiment.

SummaryAnsamitocins are potent antitumor maytansinoids produced by Actinosynnema pretiosum. Their biosynthesis involves the initial assembly of a macrolactam polyketide, followed by a series of postpolyketide synthase (PKS) modifications. Three ansamitocin glycosides were isolated from A. pretiosum and fully characterized structurally as novel ansamitocin derivatives, carrying a β-D-glucosyl group attached to the macrolactam amide nitrogen in place of the N-methyl group. By gene inactivation and complementation, asm25 was identified as the N-glycosyltransferase gene responsible for the macrolactam amide N-glycosylation of ansamitocins. Soluble, enzymatically active Asm25 protein was obtained from asm25-expressing E. coli by solubilization from inclusion bodies. Its optimal reaction conditions, including temperature, pH, metal ion requirement, and Km/Kcat, were determined. Asm25 also showed broad substrate specificity toward other ansamycins and synthetic indolin-2-ones. To the best of our knowledge, this represents the first in vitro characterization of a purified antibiotic N-glycosyltransferase.

Feeding experiments with isotope-labeled precursors rule out hydroxypyruvate and TCA cycle intermediates as the metabolic source of methoxymalonyl-ACP, the substrate for incorporation of "glycolate" units into ansamitocin P-3, soraphen A, and other antibiotics. They point to 1,3-bisphosphoglycerate as the source of the methoxymalonyl moiety and show that its C-1 gives rise to the thioester carbonyl group (and hence C-1 of the "glycolate" unit), and its C-3 becomes the free carboxyl group of methoxymalonyl-ACP, which is lost in the subsequent Claisen condensation on the type I modular polyketide synthases (PKS). d-[1,2-(13)C(2)]Glycerate is also incorporated specifically into the "glycolate" units of soraphen A, but not of ansamitocin P-3, suggesting differences in the ability of the producing organisms to activate glycerate. A biosynthetic pathway from 1,3-bisphosphoglycerate to methoxymalonyl-ACP is proposed. Two new syntheses of R- and S-[1,2-(13)C(2)]glycerol were developed as part of this work.

Because of their ecological functions, natural products have been optimized in evolution for interaction with biological systems and receptors. However, they have not necessarily been optimized for other desirable drug properties and thus can often be improved by structural modification. Using examples from the literature, this paper reviews the opportunities for increasing structural diversity among natural products by combinatorial biosynthesis, i.e., the genetic manipulation of biosynthetic pathways. It distinguishes between combinatorial biosynthesis in a narrower sense to generate libraries of modified structures, and metabolic engineering for the targeted formation of specific structural analogs. Some of the problems and limitations encountered with these approaches are also discussed. Work from the author’s laboratory on ansamycin antibiotics is presented which illustrates some of the opportunities and limitations.

SummaryA 45 kb DNA sequencing analysis from Streptomyces hygroscopicus 5008 involved in validamycin A (VAL-A) biosynthesis revealed 16 structural genes, 2 regulatory genes, 5 genes related transport, transposition/integration or tellurium resistance; another 4 genes had no obvious identity. The VAL-A biosynthetic pathway was proposed, with assignment of the required genetic functions confined to the sequenced region. A cluster of eight reassembled genes was found to support VAL-A synthesis in a heterologous host, S. lividans 1326. In vivo inactivation of the putative glycosyltransferase gene (valG) abolished the final attachment of glucose for VAL production and resulted in accumulation of the VAL-A precursor, validoxylamine, while the normal production of VAL-A could be restored by complementation with valG. The role of valG in the glycosylation of validoxylamine to VAL-A was demonstrated in vitro by enzymatic assay.

Maytansine, a potent clinically evaluated plant-derived anti-tumor drug, and its microbial counterpart, ansamitocin P-3, showed a substantially higher cytoxicity than many other anti-tumor drugs. Owing to a shortage of material and lack of sufficiently sensitive analytical methods at the time, no metabolism studies were apparently carried out in conjunction with the initial preclinical and clinical studies on maytansine, but some products of decomposition during the period of storage of the formulated drug were reported. In the current study, the in vitro metabolism of maytansine and ansamitocin P-3 was studied after incubation with rat and human liver microsomes in the presence of NADPH, and with rat and human plasma and whole blood, using liquid chromatography/multi-stage mass spectrometry. Unchanged ansamitocin P-3 and 11 metabolites and unchanged maytansine and seven metabolites were profiled and the structures of some metabolites were tentatively assigned based on their multi-stage electrospray ion-trap mass fragmentation data and in some cases accurate mass measurement. The major pathway of ansamitocin P-3 metabolism in human liver microsomes appears to be demethylation at C-10. Oxidation and sequential oxidation/demethylation also occurred, although to a lesser extent. However, the major pathway of maytansine metabolism in human liver microsomes is N-demethylation of the methylamide of the ester moiety. Several minor pathways including O/N-demethylation, oxidation and hydrolysis of the ester bond were also observed. There were no differences in maytansine metabolism between rat and human liver microsomes; however, the rate of metabolism of ansamitocin P-3 was different in rat and human liver microsomes. About 20% of ansamitocin P-3 was converted to its metabolites in rat liver microsomes and about 70% in human liver microsomes under the same conditions. Additionally, 10-O-demethylated ansamitocin P-3 was also detected in the urine after i.v. bolus administration of ansamitocin P-3 to Sprague-Dawley male rats. No metabolites were detected following incubation of maytansine and ansamitocin P-3 with human and rat whole blood and plasma.

Ansamitocin P-3 (AP3) is a potent maytansinoid antitumor agent isolated from microorganisms and mosses. In this study, a highly sensitive and specific electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for quantitation of AP3 was developed and validated. AP3 was extracted from rat plasma along with the internal standard, depsipeptide FK228 (NSC-630176, FR) with ethyl acetate. Components in the extract were separated on a 50 mm × 2.1 mm Betabasic C 85 μm stainless steel column by isocratic elution with 70% acetonitrile/0.9% formic acid. The liquid flow was passed through a pre-source splitter and 5% of the eluent was introduced into the API source. The components were analyzed in the multiple-reaction-monitoring (MRM) mode as the precursor/product ion pair of m / z 635.2/547.2 for AP3 and of m / z 541.5/424.0 for the internal standard FR. Linear calibration curves were obtained in the range 1–500 ng/mL using 0.2 mL rat plasma. The within-day coefficients of variation (CVs) were 12.9, 6.7, and 5.5% and the between-day CVs were 10.4, 6.5, and 6.4% (all n = 5) at 1, 10, and 200 ng/mL, respectively. A formulation based on normal saline and PEG300 was then developed and Sprague–Dawley male rats were given this formulated drug by i.v. bolus. Plasma drug concentrations were measured by this method and the pharmacokinetics were analyzed by standard techniques. Plasma concentration–time profiles were found to follow a triexponential decline and the terminal phase was nearly flat, suggesting that the drug distributed in deep tissue compartments or organs and then equilibrates slowly with the blood stream.

Asukamycin (2), a metabolite of Streptomyces nodosus ssp. asukaensis ATCC 29757 and a member of the manumycin family of antibiotics, is assembled from three components, an "upper" polyketide chain initiated by cyclohexanecarboxylic acid, a "lower" polyketide chain initiated by the novel starter unit, 3-amino-4-hydroxybenzoic acid (3,4-AHBA), and a cyclized 5-aminolevulinic acid moiety, 2-amino-3-hydroxycyclopent-2-enone (C(5)N unit). To shed light on the order in which these components are assembled, we synthesized in labeled form various potential intermediates and evaluated their incorporation into 2. The assembly of the molecular framework of 2 from 3,4-AHBA and cyclohexanecarboxylic acid apparently does not involve free, unactivated intermediates. However, protoasukamycin (12), the total synthesis of which is reported, was efficiently converted into 2, demonstrating that the modification of the aromatic ring to the epoxyquinol structure is the terminal step in the biosynthesis. The results suggest that the two polyketide chains are synthesized separately and that the "upper" chain must be connected to the "lower" polyketide chain before the C(5)N unit.

Maytansine and its congeners have been isolated from higher plants, mosses and from an Actinomycete, Actinosynnema pretiosum. Many of these compounds are antitumor agents of extraordinary potency, yet phase II clinical trials with maytansine proved disappointing. The chemistry and biology of maytansinoids has been reviewed repeatedly in the late 1970s and early 1980s; the present review covers new developments in this field during the last two decades. These include the use of maytansinoids as "warheads" in tumor-specific antibodies, preliminary metabolism studies, investigations of their biosynthesis at the biochemical and genetic level, and ecological issues related to the occurrence of such typical microbial metabolites in higher plants.

Maytansinoids are potent antitumor agents found in plants and microorganisms. To elucidate their biosynthesis at the biochemical and genetic level and to set the stage for their structure modification through genetic engineering, we have cloned two gene clusters required for the biosynthesis of the maytansinoid, ansamitocin, from a cosmid library of Actinosynnema pretiosum ssp. auranticum ATCC 31565. This is a rare case in which the genes involved in the formation of a secondary metabolite are dispersed in separate regions in an Actinomycete. A set of genes, asm22–24 , asm43–45 , and asm47 , was identified for the biosynthesis of the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA). Remarkably, there are two AHBA synthase gene homologues, which may have different functions in AHBA formation. Four type I polyketide synthase genes, asmA–D , followed by the downloading asm9 , together encode eight homologous sets of enzyme activities (modules), each catalyzing a specific round of chain initiation, elongation, or termination steps, which assemble the ansamitocin polyketide backbone. Another set of genes, asm13–17 , encodes the formation of an unusual “methoxymalonate” polyketide chain extension unit that, notably, seems to be synthesized on a dedicated acyl carrier protein rather than as a CoA thioester. Additional ORFs are involved in postsynthetic modifications of the initial polyketide synthase product, which include methylations, an epoxidation, an aromatic chlorination, and the introduction of acyl and carbamoyl groups. Tentative functions of several asm genes were confirmed by inactivation and heterologous expression.

Echinosporin (1), a known antibiotic with a unique tricyclic acetal-lactone structure, is produced by Streptomyces erythraeus (strain Tu 4015), together with the novel 7-deoxyechinosporin (2) as minor compound, which was fully characterized. The biosynthesis of 1 was established by feeding experiments with 13C-labelled precursors. The results revealed that 1 was formed by the shikimate pathway, with chorismate as a biosynthetic intermediate. The proposed mechanism for the conversion of chorismate into 1 represents a new branch of the shikimate pathway, producing a nonaromatic metabolite. In addition, the influence of aromatic amino acids and of glyphosate, an inhibitor in the shikimate pathway of plants, was investigated. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

To study the biosynthesis of the pseudotetrasaccharide acarbose, dTDP-4-amino-4,6-dideoxy-α- d -glucose ( 3 ) was prepared from galactose in 16 steps. After initial protecting-group manipulations, the 6-position of galactose was deoxygenated by hydride displacement of a tosylate. Similarly a tosyl group at the 4-position was displaced upon reaction with sodium azide. Conversion of the resulting glycoside to a glycosyl phosphate was accomplished by reaction of a glycosyl trichloroacetimidate with dibenzyl phosphate. Subsequent removal of the benzyl protecting groups and reduction of the azide by hydrogenation and coupling with an activated nucleoside phosphate gave dTDP-4-amino-4,6-dideoxy-α- d -glucose.

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.

The biosynthesis of ansamycin antibiotics, including rifamycin B, involves the synthesis of an aromatic precursor, 3-amino-5-hydroxybenzoic acid (AHBA), which serves as starter for the assembly of the antibiotics' polyketide backbone. The terminal enzyme of AHBA formation, AHBA synthase, is a dimeric, pyridoxal 5'-phosphate (PLP) dependent enzyme with pronounced sequence homology to a number of PLP enzymes involved in the biosynthesis of antibiotic sugar moieties. The structure of AHBA synthase from Amycolatopsis mediterranei has been determined to 2.0 A resolution, with bound cofactor, PLP, and in a complex with PLP and an inhibitor (gabaculine). The overall fold of AHBA synthase is similar to that of the aspartate aminotransferase family of PLP-dependent enzymes, with a large domain containing a seven-stranded beta-sheet surrounded by alpha-helices and a smaller domain consisting of a four-stranded antiparallel beta-sheet and four alpha-helices. The uninhibited form of the enzyme shows the cofactor covalently linked to Lys188 in an internal aldimine linkage. On binding the inhibitor, gabaculine, the internal aldimine linkage is broken, and a covalent bond is observed between the cofactor and inhibitor. The active site is composed of residues from two subunits of AHBA synthase, indicating that AHBA synthase is active as a dimer.

The biosynthetic pathway leading to the mC7N cyclitol (valienamine) moiety of acarbose (1) in Actinoplanes sp. strain SN 223/29 has been studied using 3H-, 2H-, and 13C-labeled cyclitols. These pre...

Stable isotope labeling experiments have shown that the biosynthesis of the monomeric phenazines, the saphenyl esters, and their dimerization products, the esmeraldins, in Streptomyces antibioticus Tu 2706 proceeds from phenazine-1,6-dicarboxylic acid by chain extension with C-2 of acetate to 6-acetylphenazine-1-carboxylic acid, which is reduced to saphenic acid. The latter is incorporated into both halves of the esmeraldins, albeit differentially. By feeding of chiral acetate, degradation of the resulting saphenyl esters and esmeraldins, and configurational analysis of the acetic acid formed, the chain extension process was found to proceed with overall inversion of configuration at the methyl group. This suggests that the decarboxylation of a hypothetical intermediate β-keto acid proceeds in an inversion mode. This result is discussed with reference to analogous C-methylations of polyketide backbones by addition of C-2 of acetate.

The single recombinant expressing the Streptomyces coelicolor minimal whiE (spore pigment) polyketide synthase (PKS) is uniquely capable of generating a large array of well more than 30 polyketides, many of which, so far, are novel to this recombinant. The characterized polyketides represent a diverse set of molecules that differ in size (chain length) and shape (cyclization pattern). This combinatorial biosynthetic library is, by far, the largest and most complex of its kind described to date and indicates that the minimal whiE PKS does not independently control polyketide chain length nor dictate the first cyclization event. Rather, the minimal PKS enzyme complex must rely on the stabilizing effects of additional subunits (i.e., the cyclase whiE -ORFVI) to ensure that the chain reaches the full 24 carbons and cyclizes correctly. This dramatic loss of control implies that the growing polyketide chain does not remain enzyme bound, resulting in the spontaneous cyclization of the methyl terminus. Among the six characterized dodecaketides, four different first-ring cyclization regiochemistries are represented, including C7/C12, C8/C13, C10/C15, and C13/C15. The dodecaketide TW93h possesses a unique 2,4-dioxaadamantane ring system and represents a new structural class of polyketides with no related structures isolated from natural or engineered organisms, thus supporting the claim that engineered biosynthesis is capable of producing novel chemotypes.

The enzymatic synthesis of isotopically labeled d -sedoheptulose 7-phosphate from either labeled d -glucose or labeled l -serine is described. [7- 14 C , 7- 3 H ] Sedoheptulose 7-phosphate is prepared with transketolase from xylulose 5-phosphate, as C 2 donor, and d - [5- 14 C , 5- 3 H ] ribose 5-phosphate, which in turn is generated from d - [6- 14 C , 6- 3 H ] glucose with the glycolytic enzymes. [1- 13 C ] Sedoheptulose 7-phosphate is obtained in a one-pot reaction in 69% yield from unlabeled ribose 5-phosphate and l - [3- 13 C ] serine via hydroxypyruvate as the C 2 donor using the enzymes alanine racemase, d -amino acid oxidase, catalase and transketolase. By the latter route labeled [1- 13 C ] ido -heptulose 7-phosphate was also prepared when xylose 5-phosphate was substituted for the ribose 5-phosphate.

A series of 12 recombinants expressing sets of polyketide synthase (PKS) genes from the whiE (Streptomyces coelicolor), sch (S. halstedii), and cur (S. curacoi) spore pigment biosynthetic gene clusters were prepared and shown to produce four groups of novel polyketides. Mixtures of undecaketides and dodecaketides were produced by the minimal PKS alone (TW93b, TW93c, and TW93d) or in the presence of the (unnatural) act ketoreductase (KR) (TW94b, TW94c, and TW94d), whereas when the whiE-ORFVI cyclase was present, only dodecaketides (TW95a and TW95b) arose, in high yield. This implies that the whiE minimal PKS requires an additional subunit (the cyclase) to stabilize the complex between the long nascent polyketide chain and the minimal PKS to ensure that the chain reaches the full 24 carbons. These experiments suggest that the native spore pigment is a C24 molecule with a pentacenequinone structure which is first cyclized C9 to C14. A fourth set of uncharacterized polyketides was produced when the complete s...

In preparation for biosynthetic studies on the 3,4-dihydroxy-2, 6-dimethyl-5-phenylvaleric acid portion of neoantimycin (1), the 1H and 13C NMR signals of 1 were assigned unambiguously by means of 2D correlation spectroscopy and NOE experiments. The previously undetermined absolute stereochemistry at C-15 and C-16 was deduced as (S) and (S). The structure of isoneoantimycin (2) was also elucidated. The methyl groups of methionine and propionate were incorporated stereospecifically into C-13 and C-12 of 1, respectively, and the configuration of the methyl group of methionine is inverted in the process. The results also suggest the intervention of phenylpyruvate as an actual precursor.

The biosynthesis of ansamycin antibiotics, like rifamycin B, involves formation of 3-amino-5-hydroxybenzoic acid (AHBA) by a novel variant of the shikimate pathway. AHBA then serves as the starter unit for the assembly of a polyketide which eventually links back to the amino group of AHBA to form the macrolactam ring. The terminal enzyme of AHBA formation, which catalyzes the aromatization of 5-deoxy-5-amino-3-dehydroshikimic acid, has been purified to homogeneity from Amycolatopsis mediterranei, the encoding gene has been cloned, sequenced, and overexpressed in Escherichia coli. The recombinant enzyme, a (His)6 fusion protein, as well as the native one, are dimers containing one molecule of pyridoxal phosphate per subunit. Mechanistic studies showed that the enzyme-bound pyridoxal phosphate forms a Schiff's base with the amino group of 5-deoxy-5-amino-3-dehydroshikimic acid and catalyzes both an alpha, beta-dehydration and a stereospecific 1,4-enolization of the substrate. Inactivation of the gene encoding AHBA synthase in the A. mediterranei genome results in loss of rifamycin formation; production of the antibiotic is restored when the mutant is supplemented with AHBA.

Reaction of DNA with the carcinogens N-methyl-N-nitrosourea and N-nitroso-N,N-dimethylamine produces several methylated species including the premutagenic O6-methylguanine. The mechanism of methylation is believed to be through a methanediazonium ion. We have studied the mechanism of methylation of DNA by these carcinogens by analyzing the stereochemistry of the methyl transfer. DNA was methylated in vitro by (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea and in vivo by (R)- and (S)-N-[2H1,3H]methyl-N-methyl-N-nitrosamine and (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea. 7-Methylguanine, 3-methyladenine, O6-methylguanine, and the methylated phosphate backbone were isolated. The methyl groups were converted into acetic acid, and the stereochemistry was analyzed. The identity of the nucleophile did not influence the stereochemistry of the methylation reaction. It was found that the methyl group was transferred with an average of 73% inversion and 27% retention of configuration. The most likely mechanism for the retention of configuration is through multiple methylation events in which nucleophiles which initially react with the methanediazonium ion react as electrophiles with DNA.

The tritiated chiral alkanes (S)-[1-2H1,1-3H]ethane, (R)-[1-2H1,1-3H]ethane, (S)-[1-2H1,1-3H]butane, (R)-[1-2H1,1-3H]butane, (S)-[2-3H]butane, (R)-[2-3H]butane, and racemic [2-3H]butane were oxidized by soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), and the absolute stereochemistry of the resulting product alcohols was determined in order to probe the mechanism of substrate hydroxylation. When purified hydroxylase, coupling protein, and reductase components were used, the product alcohol displayed 72% retention of stereochemistry at the labeled carbon for the ethane substrates and 77% retention for the butanes labeled at the primary carbon. A putative alkyl radical which would yield these product distributions would have a lifetime of 100 fs, a value too short to correspond to a discrete intermediate. Intramolecular kH/kD ratios of 3.4 and 2.2 were determined for ethane and butane, respectively. When the hydroxylations were performed with purified hydroxylase but only a partial...

Specifically 13C-labeled quinoline-2-carboxylate derivatives were synthesized from quinoline and used to study the biosynthesis of thiostrepton in a strain of Streptomyces laurentii. 13C NMR analysis of thiostrepton recovered after feeding methyl (RS)-[11-13C]-4-(1-hydroxyethyl)quinoline-2-carboxylate or methyl [11-13C]-4-acetylquinoline-2-carboxylate showed conclusively that these compounds are specifically and efficiently incorporated into thiostrepton. Both compounds were also detected in cultures of the producing organism by isotope dilution analysis. The significance of the relative endogenous concentrations of the two compounds and of the relative extent of the incorporation of exogenously added labeled material into thiostrepton are discussed in terms of the biosynthetic pathway linking tryptophan and 4-(1-hydroxyethyl)quinoline-2-carboxylate in S. laurentii. A highly specific enzyme activity was detected in cell-free extracts of S. laurentii that was capable of adenylating (12S)-4-(1-hydroxyethyl)quinoline-2-carboxylic acid. Partial purification of the enzyme was achieved. The enzyme was found to be specific for the enantiomer of the substrate which has the same absolute configuration as found in the natural antibiotic structure. The presence of one specific enzyme catalysing the adenylation process in S. laurentii was shown by photoaffinity labeling with [alpha-32P]-8-azido-ATP and subsequent SDS PAGE analysis of the labeled products. The native molecular weight of the active enzyme, determined by gel permeation chromatography, was found to be approximately 47 kDa, compared with a denatured weight of 50 kDa estimated for the photoaffinity-labeled protein. The enzyme is thus probably monomeric.

The biosynthetic pathway of 3-amino-5-hydroxybenzoic acid (AHBA) formation was studied with cell-free extracts from the rifamycin B producer, Amycolatopsis mediterranei S699, and the ansatrienin A producer, Streptomyces collinus Tu1892. Phosphoenolpyruvate (PEP) plus erythrose 4-phosphate (E4P) gave AHBA in low but nevertheless significant (6%) yield. 3,4-Dideoxy-4-amino-d-arabino-heptulosonic acid 7-phosphate (aminoDAHP) was converted efficiently into AHBA (45%), as were 5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ, 41%) and 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS, 95%). On the other hand, the normal shikimate pathway intermediate, 3-deoxy-d-arabino-heptulosonic acid 7-phosphate (DAHP) did not give rise to AHBA under these conditions. AminoDAHP (9%) was produced by incubation of [14C]PEP and E4P, but not of [14C]DAHP, with the cell-free extracts. The results demonstrate the operation of a new variant of the shikimate pathway in the formation of the mC7N units of ansamycin, and presumably also ...

The first committed step in the formation of taxol has been shown to involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The formation of this endocyclic diterpene olefin isomer as the precursor of taxol was unexpected, since the exocyclic isomer, taxa-4(20),11(12)-diene, had been predicted as the initial product of the taxol pathway on the basis of metabolite co-occurrence. [1-2H2,20-2H3] and [20-2H3]geranylgeranyl diphosphates were employed as substrates with the partially purified taxadiene synthase from Pacific yew (Taxus brevifolia) stems to examine the possibility of a preliminary cyclization to taxa-4(20),11(12)-diene followed by isomerization to the more stable endocyclic double bond isomer. GLC-MS analysis of the derived taxa-4(5),11(12)-diene, via selected ion monitoring of the parent ion and the P-15 and C-ring fragment ions, compared to those of unlabeled standard, showed the olefin product to possess a deuterium enrichment essentially identical to that of the acyclic precursor, thus ruling out the putative isomerization step. With [4-2H2]geranylgeranyl diphosphate as substrate, similar product analysis established the enzymatically derived taxa-4(5),11(12)-diene to contain only one deuterium atom, consistent with direct formation from a taxenyl cation by deprotonation at C5. (+/-)-Casbene, (+/-)-verticillene, and (+/-)-taxa-4(20),11(12)-diene were tested as possible olefinic intermediates in taxa-4(5),11(12)-diene formation by a series of inhibition, trapping, and direct conversion experiments; no evidence was obtained that these exogenous olefins could serve as intermediates of the cyclization reaction. However, GLC-MS analysis of the taxadiene product derived by enzymatic cyclization of [1-3H]geranylgeranyl diphosphate in 2H2O indicated little incorporation of deuterium from the medium and suggested a rapid internal proton transfer in a tightly bound olefinic intermediate. Analysis of the enzymatic product generated from [10-2H1]geranylgeranyl diphosphate confirmed the intramolecular hydrogen transfer from C11 of a verticillyl intermediate to the C-ring of taxa-4(5),11(12)-diene. From these results, a stereochemical mechanism is proposed for the taxadiene synthase reaction involving the initial cyclization of geranylgeranyl diphosphate to a transient verticillyl cation intermediate, with transfer of the C11 alpha-proton to C7 to initiate transannular B/C-ring closure to the taxenyl cation, followed by deprotonation at C5 to yield the taxa-4(5),11(12)-diene product directly.

A common approach to identify and clone biosynthetic gene from an antibiotic-producing streptomycete is to clone the resistance gene for the antibiotic of interest and then use that gene to clone DNA that is linked to it. As a first step toward cloning the genes responsible for the biosynthesis of thiostrepton (Th) in Streptomyces laurentii (Sl), the Th resistance-encoding gene (tsnR) was cloned as a 1.5-kb BamHI-PvuII fragment in Escherichia coli (Ec), and shown to confer Th resistance when introduced into S. lividans TK24. The tsnR-containing DNA fragment was used as a probe to isolate clones from cosmid libraries of DNA in the Ec cosmid vector SuperCos, and pOJ446 (an Ec/streptomycete) cosmid vector. Sequence and genetic analysis of the DNA flanking the tsnR indicates that the Sl tsnR is not closely linked to biosynthetic genes. Instead it is located within a cluster of ribosomal protein operons.

A 50 kb region of DNA from Streptomyces violaceoruber Tü22, containing genes encoding proteins involved in the biosynthesis of granaticin, was isolated. The DNA sequence of a 7.3 kb fragment from this region, located approximately 10 kb from the genes that encode the polyketide synthetase responsible for formation of the benzoisochromane quinone skeleton, revealed five open reading frames (ORF1-ORF5). The deduced amino acid sequence of GraE, encoded by ORF2, shows 60.8% identity (75.2% similarity) to a dTDP-glucose dehydratase (StrE) from Streptomyces griseus. Cultures of Escherichia coli containing plasmids with ORF2, on a 2.1 kb BamHI fragment, were able to catalyze the formation of dTDP-4-keto-6-deoxy-D-glucose from dTDP-glucose at 5 times the rate of control cultures, confirming that ORF2 encodes a dTDP-glucose dehydratase. The amino acid sequence encoded by ORF3 (GraD) is 51.4% identical (69.9% similar) to that of StrD, a dTDP-glucose synthase from Streptomyces griseus. The amino acid sequence encoded by ORF4 shares similarities with proteins that confer resistance to tetracycline and methylenomycin, and is suggested to be involved in transporting granaticin out of the cells by an active efflux mechanism.

Alkylation-amination of the enamine 2 in the presence of ethyl α,α-bis(dibromomethyl)acetate, triethyl-amine, and methylamine lead to the construction of the aza-transposed ergoline 3. Sequential reduction, hydrolysis, reesterification, and indolization of 3, produced three diastereomers of 6. The structure of these three diastereomers was assigned on the basis of nmr and ir spectral analysis to be (α-cis) syn, (β-cis) anti, and (α-trans) syn. The isomer (β-cis) anti was reduced with lithium aluminum hydride to the corresponding alcohol.

The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-L-methionine (Km = 0.1 mM) and an RNA fragment containing nucleotides 1029-1122 of the 23S ribosomal RNA from E. coli (Km = 0.001 mM) were determined. S-Adenosyl-L-homocysteine showed competitive product inhibition (Ki = 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA fragment with mutations in nucleotides 1051-1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.

The biosynthesis of the potent marine antibiotic, pentabromopseudilin (1), was investigated in Afteromonas luteoviofaceus grown in a complex medium by feeding experiments with 13 C-acetate, various amino acids, and differently labeled glucoses. The results show that the benzene ring of 1 originates from the shikimate pathway via p-hydroxybenzoic acid as a direct precursor. No incorporation of 13 C into the pyrrole ring was observed in any of these experiments

Feeding experiments with 13 C and 18 O-labeled precursors revealed that the polycyclic xanthone antibiotics, the lysolipins X (1) and I (2), is derived from the polyketide pathway (12 malonate units), the C 1 pool (methionine), molecular oxygen, and the nitrogen pool. Surprisingly, an intact malonate moiety serves as the three-carbon starter unit of the polyketide backbone, and 9 of the 12 oxygen atoms of I originate from molecular oxygen, including both of the xanthone oxygen atoms. The orientation of the malonate unit incorporated intact into lysolipin is unique and opposite from those in tetracycline and cycloheximide, i.e., the activated carbon of malonyl CoA is bound to the nitrogen of the lysolipin isoquinoline ring and the CO 2 -derived carbon serves as the starter of the polyketide chain

The biosynthesis of the plant antitumor agent, taxol, was studied by feeding radioactive or stable isotope-labeled precursors to cut stems or to inner bark tissue of Taxus brevifolia. The labeled taxol was purified to radiochemical purity and subjected to chemical degradation or was analyzed by electrospray tandem mass spectrometry. It was demonstrated that in the plant taxol is synthesized from baccatin-111, containing the fully functionalized diterpene moiety, and a precursor of the phenylpropanoid side chain. The latter arises from phenylalanine not via cinnamic acid, but via B-phenylalanine and phenylisoserine. Benzoylation of the side chain occurs only after its attachment to the diterpene moiety. The benzoate moiety is also formed from phenylalanine via B-phenylalanine and phenylisoserine, not via cinnamic acid. Our studies focused initially on the origin of the phenylpropanoid side chain and its mode of attachment to the diterpene moiety. Feeding experiments with radiolabeled precursors were carried out with cut twigs of Taxus brevifolia to which the radioactive material was administered through the cut stem in a small volume of water. Extensive purification of the resulting taxol, first by repeated HPLC and then by co-crystallization with non-labeled carrier material was found necessary to achieve constant specific radioactivity or, in other cases, to remove traces of radioactive impurities. Under these conditions incorporation of (7-14C)benzoic acid, as its N-acetylcysteamine thioester (0.09%), and ( 13-3Hlbaccatin-III (0.12%) was obtained. Degradation of the radioactive taxol from the benzoate feeding experiment showed that 88% of the radioactivity was recovered in the side chain fragment and only 1 1 % in the baccatin-111, suggesting that under the conditions of the experiment relatively little of the diterpene moiety was synthesized de novo. Upon degradation of the taxol from the baccatin-I11 feeding experiments, all the radioactivity was recovered in the baccatin-111 moiety, none in the side chain fragment.

The biosynthesis of naphthomycin A (1) in Streptomyces collinus was studied in feeding experiments with single and multiple 13C-labeled precursors followed by 13C NMR analysis of the labeling and 13C–13C coupling patterns in the product. The results indicate that 1 is assembled via the polyketide pathway from 3-amino-5-hydroxybenzoic acid (2) as the starter unit (mC7N unit) plus seven propionate and six acetate chain extension units. 2 is synthesized via the shikimate pathway by a process that attaches the nitrogen to the carbon derived from C1 of erythrose 4-phosphate, consistent with a new branch of the shikimate pathway recently discovered to operate in the biosynthesis of the mC7N unit of rifamycin B.

This report demonstrates genetic transformation of two Taxus species. Taxus brevifolia and Taxus baccata, and expression of bacterial genes transferred into the plant genome by Agrobacterium tumefaciens. We used two strains of Agrobacterium tumefaciens (Bo542 and C58) to inoculate shoot segments of mature yew trees. The highest gall formation frequency (28.3%) was achieved with Taxus baccata using the Bo542 strain. Agrobacterium tumefaciens strain Bo542 induced significantly more galls (24%) than strin C58 (4%). Although we were able to induce on both Taxus species, Taxus baccata showed significantly higher susceptibility (14%) than Taxus brevifolia (7%). In contrast to untransformed callus cultures, the gall cell lines proliferated on phytormone-free medium and produced agropine as the result of T-DNA transfer. Southern blot analysis showed the presence of T-DNA sequence in the genome of these cell lines. Taxol and related taxane produced by the transgenic callus cultures were identified by mass spectrometry and immunoassay with monoclonal antibodies specific for taxol.

β-Chloro-D-alanine and D-serine O-sulfate are converted into a putative aminoacrylate intermediate by D-amino acid aminotransferase. This either reacts with pyridoxal phosphate to form a reactive inhibitor of the enzyme or it is protonated and hydrolysed to give pyruvate. The protonation reaction is shown to occur with modest stereoselectivity, indicating overall retention of stereochemistry in replacement of the leaving group by hydrogen. The corresponding reaction of L-serine O-sulfate using L-aspartate aminotransferase shows little or no stereosetectivity.

The biosynthesis of the antibiotic reductiomycin (I) in Streptomyces xanthochromogenus was investigated by feeding experiments with radioactive and stable isotope-labeled precursors. NMR and mass spectroscopic analyses of the labeled I samples revealed that the acetoxy group comes from acetate, the 2-amino-3-hydroxycyclopent-2-enone moiety arises by a novel intramolecular cyclization of 5-aminolevulinic acid (ALA), and the dihydrofuranylacrylic moiety is formed by aromatic ring cleavage of a symmetrical product of the shikimate pathway. Both 4-hydroxy-[7-[sup 13]C]benzoic acid and 4-hydroxy-[7-[sup 13]C]benzaldehyde label 1 very efficiently, and deuterium from various positions in these precursors is incorporated into the predicted positions in the dihydrofuranylacrylic acid moiety of 1. The results are interpreted in terms of a dioxygenase mechanism for the ring cleavage reaction and pyridoxal phosphate catalysis for the ALA cyclization. 42 refs., 6 figs., 5 tabs.

The biosynthesis of the phenazine antibiotics, the esmeraldins and the related saphenamycins, was studied by feeding radioactive and stable isotope-labeled precursors to Streptomyces antibioticus, strain Tu 2706. After purification, the labeled antibiotics were degraded to the diastereomeric esmeraldic acid dimethyl esters 3 and 4 and racemic saphenic acid methyl ester (5), respectively. The 1 H- and 13 C-NMR spectra of these were assigned by single and multiple bond correlation experiments

The biosynthesis of the thiopeptide antibiotic thiostrepton (1) has been investigated by administration of isotopically labeled precursors to cultures of Streptomyces azureus and Streptomyces laurentii. The amino acid origin of all the components of the antibiotic was demonstrated. Experiments with (S)-[1,2- 13 C 2 ]- and (S)-[2,3- 13 C 2 ]serine showed intact incorporation of serine into the thiazoline and thiazole rings as well as the dehydroalanine, alanine, and tetrahydropyridine moieties. (S)-[3- 13 C, 2 H 2 ]Serine and (2S,3S)-[3- 13 C, 2 H 1 ]serine were used to elucidate the stereochemistry of the various transformations of serine

The biosynthesis of the highly modified thiopeptide antibiotic, nosiheptide (1), was studied by feeding radioactive and stable-isotope-labeled precursors to cultures of the producing organism, Streptomyces actuosus. The stable isotope enrichments and/or coupling patterns in the isolated 1 were analyzed by NMR spectroscopy using various 1D and 2D NMR techniques. The results complete the confirmation of the amino acid components of the antibiotic and shed light on a number of mechanistic and stereochemical aspects of its assembly from these basic building blocks. The dehydroalanine and butyrine moieties are formed by an anti elimination of water from serine and threonine, respectively, the thiazole rings from cysteine residues with loss of the pro-3R hydrogen in the oxidation step, and the hydroxypyridine moiety from two intact serine residues, situated nine amino acids apart in the peptide chain, and the carboxyl group of an adjacent cysteine. According to {sup 15}N studies which included the complete assignment of the {sup 15}N NMR resonances of 1, the carboxy-terminal amide nitrogen of 1 originates from the amino group of serine, suggesting that the precursor peptide giving rise to 1 carries an additional carboxyl-terminal serine residue which, except for its nitrogen, is removed during processing. 64 refs., 7 figs.,more » 3 tabs.« less