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1.
Org Lett ; 22(1): 150-154, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31829601

RESUMO

A four-enzyme catalyzed hydroxy regioisomerization of anthracycline was integrated into the biosynthetic pathway of aclacinomycin A (ALM-A), to generate a series of iso-ALMs via directed combinatorial biosynthesis combined with precursor-directed mutasynthesis. Most of the newly acquired iso-ALMs exhibit obviously (1-5-fold) improved antitumor activity. Therefore, we not only developed iso-ALMs with potential as clinical drugs but also demonstrated the utility of this tailoring tool for modification of anthracycline antibiotics in drug discovery and development.


Assuntos
Aclarubicina/análogos & derivados , Antibióticos Antineoplásicos/farmacologia , Policetídeo Sintases/metabolismo , Aclarubicina/biossíntese , Aclarubicina/química , Aclarubicina/farmacologia , Antibióticos Antineoplásicos/biossíntese , Antibióticos Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Conformação Molecular , Streptomyces/química , Streptomyces/metabolismo
2.
J Am Chem Soc ; 129(34): 10546-50, 2007 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-17685523

RESUMO

The tetracyclic core of anthracycline natural products with antitumor activity such as aclacinomycin A are tailored during biosynthesis by regioselective glycosylation. We report the first synthesis of TDP-L-rhodosamine and demonstrate that the glycosyltransferase AknS transfers L-rhodosamine to the aglycone to initiate construction of the side-chain trisaccharide. The partner protein AknT accelerates AknS turnover rate for L-rhodosamine transfer by 200-fold. AknT does not affect the Km but rather affects the kcat. Using these data, we propose that AknT causes a conformational change in AknS that stabilizes the transition state and ultimately enhances transfer. When the subsequent glycosyltransferase AknK and its substrate TDP-L-fucose are also added to the aglycone, the disaccharide and low levels of a fully reconstituted trisaccharide form of aclacinomycin are observed.


Assuntos
Aclarubicina/biossíntese , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Hexosaminas/metabolismo , Macrolídeos/química , Aclarubicina/química , Aclarubicina/metabolismo , Antineoplásicos/química , Glicosilação , Cinética , Estrutura Molecular , Especificidade por Substrato
3.
Proc Natl Acad Sci U S A ; 104(15): 6170-5, 2007 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-17395717

RESUMO

Aclacinomycin (Acl) oxidoreductase (AknOx) catalyzes the last two steps in the biosynthesis of polyketide antibiotics of the Acl group, the oxidation of the terminal sugar moiety rhodinose to l-aculose. We present the crystal structure of AknOx with bound FAD and the product AclY, refined to 1.65-A resolution. The overall fold of AknOx identifies the enzyme as a member of the p-cresol methylhydroxylase superfamily. The cofactor is bicovalently attached to His-70 and Cys-130 as 8alpha-Ndelta1-histidyl, 6-S-cysteinyl FAD. The polyketide ligand is bound in a deep cleft in the substrate-binding domain, with the tetracyclic ring system close to the enzyme surface and the three-sugar chain extending into the protein interior. The terminal sugar residue packs against the isoalloxazine ring of FAD and positions the carbon atoms that are oxidized close to the N5 atom of FAD. The structure and site-directed mutagenesis data presented here are consistent with a mechanism where the two different reactions of AknOx are catalyzed in the same active site but by different active site residues. Tyr-450 is responsible for proton removal from the C-4 hydroxyl group in the first reaction, the oxidation of rhodinose to cinerulose A. Tyr-378 acts as a catalytic base involved in proton abstraction from C3 of cinerulose A in the second reaction, for formation L-aculose. Replacement of this residue, however, does not impair the conversion of rhodinose to cinerulose A.


Assuntos
Aclarubicina/análogos & derivados , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Modelos Moleculares , Streptomyces/enzimologia , Aclarubicina/biossíntese , Sequência de Aminoácidos , Catálise , Biologia Computacional , Cristalografia , Escherichia coli , Dados de Sequência Molecular , Estrutura Molecular , Monossacarídeos/metabolismo , Mutagênese Sítio-Dirigida , Conformação Proteica
4.
J Am Chem Soc ; 127(35): 12254-62, 2005 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-16131203

RESUMO

Aklanonic acid, an anthraquinone natural product, is a common advanced intermediate in the biosynthesis of several antitumor polyketide antibiotics, including doxorubicin and aclacinomycin A. Intensive semisynthetic and biosynthetic efforts have been directed toward developing improved analogues of these clinically important compounds. The primer unit of such polyfunctional aromatic polyketides is an attractive site for introducing novel chemical functionality, and attempts have been made to modify the primer unit by precursor-directed biosynthesis or protein engineering of the polyketide synthase (PKS). We have previously demonstrated the feasibility of engineering bimodular aromatic PKSs capable of synthesizing unnatural hexaketides and octaketides. In this report, we extend this ability by preparing analogues of aklanonic acid, a decaketide, and its methyl ester. For example, by recombining the R1128 initiation module with the dodecaketide-specific pradimicin PKS, the isobutyryl-primed analogue of aklanonic acid (YT296b, 10) and its methyl ester (YT299b, 12) were prepared. In contrast, elongation modules from dodecaketide-specific spore pigment PKSs were unable to interact with the R1128 initiation module. Thus, in addition to revealing a practical route to new anthracycline antibiotics, we also observed a fundamental incompatibility between antibiotic and spore pigment biosynthesis in the actinomycetes bacteria.


Assuntos
Antraquinonas/metabolismo , Antibióticos Antineoplásicos/biossíntese , Biotecnologia , Aclarubicina/biossíntese , Actinomycetales/metabolismo , Antraciclinas/farmacologia , Antraquinonas/química , Antibióticos Antineoplásicos/farmacologia , Sequência de Bases , Sítios de Ligação , Daunorrubicina/biossíntese , Doxorrubicina/biossíntese , Ésteres/química , Ésteres/metabolismo , Macrolídeos/metabolismo , Dados de Sequência Molecular , Policetídeo Sintases/metabolismo
5.
Biochemistry ; 43(15): 4548-58, 2004 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-15078101

RESUMO

The antitumor drug aclacinomycin A is a representative member of the anthracycline subgroup that contains a C(7)-O-trisaccharide chain composed of L-2-deoxysugars. The sugar portion of the molecule, which greatly affects its biological activity, is assembled by dedicated glycosyltransferases; however, these enzymes have not been well-studied. Here we report the heterologous expression and purification of one of these enzymes, AknK, as well as the preparation of dTDP-L-2-deoxysugar donors, dTDP-L-2-deoxyfucose and dTDP-L-daunosamine, and the monoglycosyl aglycone, rhodosaminyl aklavinone. Our experiments show that AknK catalyzes the addition of the second sugar to the chain, using dTDP-L-2-deoxyfucose and rhodosaminyl aklavinone, to create the L-2-deoxyfucosyl-L-rhodosaminyl aklavinone. AknK also accepts an alternate dTDP-L-sugar, dTDP-L-daunosamine, and other monoglycosylated anthracyclines, including daunomycin, adriamycin, and idarubicin, to build alternate disaccharides on variant anthracycline backbones. Remarkably, AknK also catalyzes a tandem addition of a second L-2-deoxyfucosyl moiety, albeit with reduced activity, to the natural disaccharide chain to produce L-deoxyfucosyl-L-deoxyfucosyl-L-rhodosaminyl aklavinone, a variant of the natural aclacinomycin A. These results demonstrate that AknK may be a useful enzyme for the chemoenzymatic synthesis of anthracycline variants.


Assuntos
Aclarubicina/biossíntese , Fucosiltransferases/isolamento & purificação , Streptomyces/enzimologia , Aclarubicina/análogos & derivados , Catálise , Clonagem Molecular , Fucose/análogos & derivados , Fucose/biossíntese , Fucose/química , Fucosiltransferases/biossíntese , Fucosiltransferases/genética , Glicosilação , Hexosaminas/biossíntese , Hexosaminas/química , Naftacenos/síntese química , Streptomyces/genética , Especificidade por Substrato , Nucleotídeos de Timina/síntese química , Trissacarídeos/química
6.
Antimicrob Agents Chemother ; 47(4): 1291-6, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12654660

RESUMO

The biosynthesis pathways of two anthracyclines, nogalamycin and aclacinomycin, were directed toward angucyclines by using an angucycline-specific cyclase, pgaF, isolated from a silent antibiotic biosynthesis gene cluster. Addition of pgaF to a gene cassette that harbored the early biosynthesis genes of nogalamycin resulted in the production of two known angucyclinone metabolites, rabelomycin and its precursor, UWM6. Substrate flexibility of pgaF was demonstrated by replacement of the nogalamycin minimal polyketide synthase genes in the gene cassette with the equivalent aclacinomycin genes together with aknE2 and aknF, which specify the unusual propionate starter unit in aclacinomycin biosynthesis. This modification led to the production of a novel angucyclinone, MM2002, in which the expected ethyl side chain was incorporated into the fourth ring.


Assuntos
Aclarubicina/análogos & derivados , Aclarubicina/biossíntese , Antibacterianos/biossíntese , Antibióticos Antineoplásicos/biossíntese , Nogalamicina/biossíntese , Streptomyces/metabolismo , Engenharia Genética , Família Multigênica
7.
Mol Gen Genet ; 264(1-2): 164-72, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11016846

RESUMO

We have cloned and characterized a gene cluster for anthracycline biosynthesis from Streptomyces galilaeus. This cluster, 15-kb long, includes eight genes involved in the deoxyhexose biosynthesis pathway, a gene for a glycosyltransferase and one for an activator, as well as two genes involved in aglycone biosynthesis. Gene disruption targeted to the activator gene blocked production of aclacinomycins in S. galilaeus. Plasmid pSgs4, containing genes for a glycosyltransferase (aknS), an aminomethylase (aknX), a glucose-1-phosphate thymidylyltransferase (akn Y) and two genes for unidentified glycosylation functions (aknT and aknV), restored the production of aclacinomycins in the S. galilaeus mutants H063, which accumulates aklavinone, and H054, which produces aklavinone with rhodinose and deoxyfucose residues. Furthermore, pSgs4 directed the production of L-rhamnosyl-epsilon-rhodomycinone and L-daunosaminyl-epsilon-rhodomycinone in S. peucetius strains that produce epsilon-rhodomycinone endogenously. Subcloning of the gene cluster was carried out in order to further define the genes that are responsible for complementation and hybrid anthracycline generation.


Assuntos
Aclarubicina/metabolismo , Proteínas de Bactérias/genética , Família Multigênica , Streptomyces/genética , Streptomyces/metabolismo , Aclarubicina/biossíntese , Antraciclinas/metabolismo , Antibióticos Antineoplásicos/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Inativação Gênica , Teste de Complementação Genética , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Células Híbridas , Metiltransferases/genética , Dados de Sequência Molecular , Mutação , Nucleotidiltransferases/genética , Plasmídeos/genética
8.
Chem Biol ; 4(10): 751-5, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9375253

RESUMO

BACKGROUND: Mithramycin, nogalamycin and aclacinomycins are aromatic polyketide antibiotics that exhibit antitumour activity. The precursors of these antibiotics are formed via a polyketide biosynthetic pathway in which acetate (for mithramycinone and nogalamycinone) or propionate (for aklavinone) is used as a starter unit and nine acetates are used as extender units. The assembly of building blocks is catalyzed by the minimal polyketide synthase (PKS). Further steps include regiospecific reductions (if any) and cyclization. In the biosynthesis of mithramycin, however, ketoreduction is omitted and the regiospecificity of the first cyclization differs from that of anthracycline antibiotics (e.g. nogalamycin and aclacinomycins). These significant differences provide a convenient means to analyze the determinants for the regiospecificity of the first cyclization step. RESULTS: In order to analyze a possible role of the minimal PKS in the regiospecificity of the first cyclization in polyketide biosynthesis, we expressed the mtm locus, which includes mithramycin minimal PKS genes, in Streptomyces galilaeus, which normally makes aclacinomycins, and the sno locus, which includes nogalamycin minimal PKS genes, in Streptomyces argillaceus, which normally makes mithramycin. The host strains are defective in the minimal PKS, but they express other antibiotic biosynthesis genes. Expression of the sno minimal PKS in the S. argillaceus polyketide-deficient strain generated mithramycin production. Auramycins, instead of aclacinomycins, accumulated in the recombinant S. galilaeus strains, suggesting that the mithramycin minimal PKS is responsible for the choice of starter unit. We also describe structural analysis of the compounds accumulated by a ketoreductase-deficient S. galilaeus mutant; spectroscopic studies on the major polyketide compound that accumulated revealed a first ring closure which is not typical of anthracyclines, suggesting an important role for the ketoreductase in the regiospecificity of the first cyclization. CONCLUSIONS: These experiments clearly support the involvement of ketoreductase and a cyclase in the regiospecific cyclization of the biosynthetic pathway for aromatic polyketides.


Assuntos
Antraciclinas/metabolismo , Proteínas de Bactérias , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Plicamicina/biossíntese , Dobramento de Proteína , Aclarubicina/análogos & derivados , Aclarubicina/biossíntese , Oxirredutases do Álcool/genética , Antraciclinas/química , Complexos Multienzimáticos/genética , Família Multigênica , Nogalamicina/biossíntese , Plicamicina/química , Streptomyces/enzimologia , Streptomyces/genética
10.
Antimicrob Agents Chemother ; 39(7): 1616-20, 1995 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7492117

RESUMO

The aklavinone 11-hydroxylase gene and two doxorubicin resistance genes cloned from Streptomyces peucetius subsp. caesius ATCC 27952 were introduced into doxorubicin-sensitive Streptomyces galilaeus ATCC 31133, an aclacinomycin producer. The doxorubicin resistance genes drrA and drrB endowed S. galilaeus with high-level resistance to doxorubicin, indicating that the resistance mechanism for doxorubicin might be different from that for aclacinomycin A. Transformation of S. galilaeus ATCC 31133 with plasmid pMC213 containing the aklavinone 11-hydroxylase gene (dnrF) resulted in the production of many red pigments. A new metabolite was purified, and the position of the newly introduced hydroxyl group was determined. This result indicated that the aklavinone 11-hydroxylase gene was stably expressed in S. galilaeus ATCC 31133 and that it gave rise to a hybrid aclacinomycin A which showed highly specific in vitro cytotoxicity against leukemia and melanoma cell lines.


Assuntos
Aclarubicina/análogos & derivados , Doxorrubicina/farmacologia , Genes Bacterianos , Streptomyces/genética , Streptomyces/metabolismo , Aclarubicina/biossíntese , Hidrocarboneto de Aril Hidroxilases/genética , Sequência de Carboidratos , Resistência Microbiana a Medicamentos , Expressão Gênica , Humanos , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Streptomyces/enzimologia , Células Tumorais Cultivadas/efeitos dos fármacos
11.
Microbiology (Reading) ; 140 ( Pt 6): 1359-65, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8081501

RESUMO

Twelve mutants of Streptomyces galilaeus (ATCC 31615) blocked in the production of aclacinomycin A, an anthracycline antibiotic with significant antitumour activity, accumulated intermediates of the biosynthesis of aclacinomycins and several anthracyclines with variant sugar moieties. Three of these aklavinone glycosides have not been described before. Mutant strains H028, H061 and H036 were blocked before the formation of aklavinone, a common intermediate for most anthracyclines. Strain H039 accumulated aklavinone and H026, H035, H038 and H054 had mutations that changed glycosylation of aklavinone. Characterization of the mutants and their products is described.


Assuntos
Aclarubicina , Aclarubicina/análogos & derivados , Streptomyces/genética , Acetatos/metabolismo , Aclarubicina/biossíntese , Antraquinonas/metabolismo , Sequência de Carboidratos , Carboidratos/química , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese , Naftacenos/metabolismo , Propionatos/metabolismo , Streptomyces/metabolismo
12.
J Antibiot (Tokyo) ; 46(8): 1219-31, 1993 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8407584

RESUMO

Microbial conversion of anthracyclinone monosaccharides using aclarubicin-negative mutant of Streptomyces galilaeus was found to produce anthracyclinone disaccharides which had either rhodinose or 2-deoxyfucose as an additional sugar. By this conversion we obtained twelve new anthracyclines from seven anthracyclines which had rhodosamine, N-monomethyldaunosamine or daunosamine at C-7 as a glycosidic sugar. All products had a reduced cytotoxic activity in comparison with those of parent compounds. However, some of them showed a therapeutically improved antitumor effects against L1210 leukemia in vivo.


Assuntos
Aclarubicina/biossíntese , Antibióticos Antineoplásicos/metabolismo , Streptomyces/metabolismo , Animais , Antibióticos Antineoplásicos/isolamento & purificação , Antibióticos Antineoplásicos/farmacologia , Glicosilação , Leucemia L1210/patologia , Mutação , Streptomyces/genética , Relação Estrutura-Atividade , Células Tumorais Cultivadas
13.
Wei Sheng Wu Xue Bao ; 31(3): 247-50, 1991 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-1862654

RESUMO

During the course of screening for new antitumor antibiotics, a new anthracycline antibiotic--aclacinomycin A was separated from the broth and mycelium of Streptomyces AC-57. The strain AC-57 was isolated from the soil collected in the Shanghai suburbs. According to its culture and physiological characteristics the producer was identified as Str. galilaeus AC-57. The broth and mycelium were extracted and treated with solvents as usual way. The aclacinomycin A was separated by silica-gel column chromatography eluted with chrolo-form-methanol. Aclacinomycin A, its aglycone and sugar components were identified by comparison of their physico-chemical and spectral data (MS, UV, IR, 1H-NMR, and 13C-NMR) with authentic compound, purified from the market sample.


Assuntos
Aclarubicina/biossíntese , Streptomyces/isolamento & purificação , Aclarubicina/química , Streptomyces/metabolismo
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