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1.
Chem Biol ; 19(9): 1116-25, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22999880

RESUMO

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 Tü 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.


Assuntos
Vias Biossintéticas/genética , Ácidos Dicarboxílicos/metabolismo , Família Multigênica/genética , Fenazinas/metabolismo , Plasmídeos/genética , Clonagem Molecular , Ácidos Dicarboxílicos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação/genética , Fenazinas/química , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Streptomyces antibioticus/enzimologia , Streptomyces antibioticus/genética , Streptomyces antibioticus/metabolismo
3.
J Am Chem Soc ; 134(3): 1673-9, 2012 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-22136518

RESUMO

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.


Assuntos
Amida Sintases/metabolismo , Benzoquinonas/metabolismo , Lactamas Macrocíclicas/metabolismo , Streptomyces/enzimologia , Amida Sintases/química , Sequência de Aminoácidos , Benzoquinonas/química , Lactamas Macrocíclicas/química , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Streptomyces/química , Especificidade por Substrato
4.
Chembiochem ; 12(11): 1759-66, 2011 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-21681880

RESUMO

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 protein encoded by asm10 belongs to the SAM-dependent methyltransferase family. Through gene inactivation and complementation, asm10 was proved to be responsible for the N-methylation of ansamitocins. Asm10 is a 33.0 kDa monomer, as determined by gel filtration. By using N-desmethyl-ansamitocin P-3 as substrate, the optimal temperature and pH for Asm10 catalysis were determined to be 32 °C and 10.0, respectively. 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 through the binding of SAM. The characterization of this unique N-methyltransferase has enriched the toolbox for engineering N-methylated derivatives from both natural and synthetic compounds; this will allow known potential drugs to be modified.


Assuntos
Amidas/metabolismo , Maitansina/análogos & derivados , Metiltransferases/metabolismo , Actinomycetales/enzimologia , Actinomycetales/metabolismo , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/metabolismo , Maitansina/biossíntese , Maitansina/química , Metilação , Metiltransferases/química , Metiltransferases/genética
5.
J Antibiot (Tokyo) ; 64(1): 35-44, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21081954

RESUMO

The aminoshikimate pathway of formation of 3-amino-5-hydroxybenzoic acid (AHBA), the precursor of ansamycin and other antibiotics is reviewed. In this biosynthesis, genes for kanosamine formation have been recruited from other genomes, to provide a nitrogenous precursor. Kanosamine is then phosphorylated and converted by common cellular enzymes into 1-deoxy-1-imino-erythrose 4-phosphate, the substrate for the formation of aminoDAHP. This is converted via 5-deoxy-5-aminodehydroquinic acid and 5-deoxy-5-aminodehydroshikimic acid into AHBA. Remarkably, the pyridoxal phosphate enzyme AHBA synthase seems to have two catalytic functions: As a homodimer, it catalyzes the last reaction in the pathway, the aromatization of 5-deoxy-5-aminodehydroshikimic acid, and at the beginning of the pathway in a complex with the oxidoreductase RifL it catalyzes the transamination of UDP-3-keto-D-glucose. The AHBA synthase gene also serves as a useful tool in the genetic screening for new ansamycins and other AHBA-derived natural products.


Assuntos
Actinomycetales/metabolismo , Aminobenzoatos/metabolismo , Hidroliases/metabolismo , Mitomicina/biossíntese , Rifabutina/metabolismo , Actinomycetales/enzimologia , Hidroxibenzoatos , Estrutura Molecular
7.
J Biol Chem ; 285(32): 24915-24, 2010 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-20522559

RESUMO

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.


Assuntos
Streptomyces/metabolismo , Antineoplásicos/farmacologia , Catálise , Química Farmacêutica/métodos , Clonagem Molecular , Desenho de Fármacos , Ácido Graxo Sintases/química , Ácidos Graxos/química , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Genéticos , Família Multigênica , Fases de Leitura Aberta , Polienos/química , Recombinação Genética , Streptomyces/enzimologia
10.
J Am Chem Soc ; 131(11): 3812-3, 2009 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-19292483

RESUMO

The timing of introduction of the unusually placed Delta(11,13) diene system in ansamitocin (AP) biosynthesis was probed by synthesizing optically active potential tri- and tetraketide intermediates as their SNAC thioesters. An AP-nonproducing mutant Actinosynnema pretiosum was complemented by the R enantiomer of the triketide and by the tetraketide with rearranged double bonds, but not by the tetraketide carrying the double bonds in conjugation to the thioester function. The results show that the double bonds are installed in their final positions during processing of the nascent polyketide on module 3 of the asm PKS and that KS4 of the PKS acts as a gatekeeper which accepts only a tetraketide with shifted double bonds as substrate for further processing.


Assuntos
Actinomycetales/metabolismo , Maitansina/análogos & derivados , Alcenos , Antibacterianos/biossíntese , Fenômenos Químicos , Maitansina/biossíntese , Moduladores de Tubulina
11.
Chem Biol ; 15(8): 863-74, 2008 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-18721757

RESUMO

Ansamitocins 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 beta-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.


Assuntos
Amidas/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Proteínas de Bactérias/metabolismo , Glucosiltransferases/metabolismo , Maitansina/análogos & derivados , Actinomycetales/enzimologia , Antifúngicos/farmacologia , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Basidiomycota/efeitos dos fármacos , Linhagem Celular Tumoral , Glicosídeos/química , Glicosídeos/metabolismo , Glicosilação , Humanos , Cinética , Lactamas/química , Lactamas/metabolismo , Maitansina/química , Maitansina/isolamento & purificação , Maitansina/metabolismo , Maitansina/farmacologia , Renaturação Proteica , Especificidade por Substrato , Uridina Difosfato Glucose/metabolismo
13.
J Am Chem Soc ; 128(44): 14325-36, 2006 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-17076505

RESUMO

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.


Assuntos
Proteína de Transporte de Acila/biossíntese , Glicolatos/química , Macrolídeos/metabolismo , Malonatos/química , Maitansina/análogos & derivados , Proteína de Transporte de Acila/química , Sequência de Aminoácidos , Isótopos de Carbono , Ciclo do Ácido Cítrico/fisiologia , Ácidos Difosfoglicéricos/química , Ácidos Difosfoglicéricos/metabolismo , Marcação por Isótopo , Macrolídeos/química , Maitansina/química , Maitansina/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Piruvatos/metabolismo
14.
Chembiochem ; 7(8): 1221-5, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16927319

RESUMO

The biosynthesis of the antitumor antibiotic, ansamitocin, involves the assembly of a linear octaketide on the ansamitocin (asm) polyketide synthase (PKS), which is then cyclized to proansamitocin and further modified to the final product. In the first chain-extension step on the asm PKS, a stereocenter is generated which is then obliterated in a subsequent double-bond migration. The cryptic configuration at this stereocenter was determined by first synthesizing the two enantiomers of the intermediate diketide as their N-acetylcysteamine (SNAC) thioesters. These were then used to demonstrate that only the R enantiomer complements a 3-amino-5-hydroxybenzoic acid (AHBA) deficient mutant of Actinosynnema pretiosum to restore ansamitocin formation. The low efficiency of complementation by the diketide, compared to AHBA, is due to inefficient loading onto the PKS and not the inhibition of the enzyme. A presumed next chain-extension intermediate-the triketide with an unrearranged double bond-was also synthesized as its SNAC ester, but did not complement the AHBA(-) mutant.


Assuntos
Actinomycetales/metabolismo , Maitansina/análogos & derivados , Policetídeo Sintases/metabolismo , Actinomycetales/genética , Maitansina/química , Maitansina/metabolismo , Estrutura Molecular , Família Multigênica , Estereoisomerismo
15.
Chem Biol ; 13(4): 387-97, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16632251

RESUMO

A 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.


Assuntos
Inositol/análogos & derivados , Streptomyces/genética , Streptomyces/metabolismo , Sequência de Bases , DNA Bacteriano/genética , Marcação de Genes , Genes Bacterianos , Genes Reguladores , Teste de Complementação Genética , Engenharia Genética , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Inositol/biossíntese , Inositol/genética , Dados de Sequência Molecular , Estrutura Molecular , Família Multigênica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces lividans/genética , Streptomyces lividans/metabolismo
16.
J Biotechnol ; 124(1): 242-57, 2006 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-16414140

RESUMO

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.


Assuntos
Técnicas de Química Combinatória , Desenho de Fármacos , Engenharia Genética/métodos , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/farmacologia , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Maitansina/síntese química , Maitansina/química , Maitansina/farmacologia , Estrutura Molecular , Policetídeo Sintases/biossíntese , Policetídeo Sintases/química , Policetídeo Sintases/genética , Policetídeo Sintases/farmacologia , Rifabutina/síntese química , Rifabutina/química , Rifabutina/farmacologia
17.
J Nat Prod ; 69(1): 158-69, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16441092

RESUMO

In this review the author traces his scientific career from its beginnings in Germany to his moves to, successively, Purdue University, The Ohio State University, and finally University of Washington. During this time his research progressed from extensive studies on ergot alkaloids, the stereochemistry of enzyme reactions, and tracer studies on antibiotic biosynthesis to its latest emphasis on the molecular biology of ansamycin antibiotics. The formative influence of several mentors and colleagues is acknowledged.


Assuntos
Produtos Biológicos , Alcaloides de Claviceps , Rifabutina , Produtos Biológicos/biossíntese , Produtos Biológicos/química , Produtos Biológicos/genética , Alcaloides de Claviceps/biossíntese , Alcaloides de Claviceps/química , Estrutura Molecular , Rifabutina/química , Rifabutina/metabolismo
18.
Appl Environ Microbiol ; 71(9): 5066-76, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16151088

RESUMO

A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Streptomyces/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Inositol/análogos & derivados , Inositol/biossíntese , Inositol/farmacologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Oryza/microbiologia , Doenças das Plantas/microbiologia , Análise de Sequência de DNA , Streptomyces/classificação , Streptomyces/genética
19.
Microbiology (Reading) ; 151(Pt 8): 2515-2528, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16079331

RESUMO

Rifamycin B biosynthesis by Amycolatopsis mediterranei S699 involves a number of unusual modification reactions in the formation of the unique polyketide backbone and decoration of the molecule. A number of genes believed to be involved in the tailoring of rifamycin B were investigated and the results confirmed that the formation of the naphthalene ring moiety of rifamycin takes place during the polyketide chain extension and is catalysed by Rif-Orf19, a 3-(3-hydroxyphenyl)propionate hydroxylase-like protein. The cytochrome P450-dependent monooxygenase encoded by rif-orf5 is required for the conversion of the Delta12, 29 olefinic bond in the polyketide backbone of rifamycin W into the ketal moiety of rifamycin B. Furthermore, Rif-Orf3 may be involved in the regulation of rifamycin B production, as its knock-out mutant produced about 40 % more rifamycin B than the wild-type. The work also revealed that many of the genes located in the cluster are not involved in rifamycin biosynthesis, but might be evolutionary remnants carried over from an ancestral lineage.


Assuntos
Actinobacteria/metabolismo , Actinobacteria/fisiologia , Macrolídeos/metabolismo , Complexos Multienzimáticos/metabolismo , Rifamicinas/biossíntese , Actinobacteria/genética , Sequência de Aminoácidos , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Rifamicinas/química
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