RESUMO
Aminoglycosides are essential antibiotics used to treat severe infections caused mainly by Gram-negative bacteria. Gentamicin is an aminoglycoside and, despite its toxicity, is clinically used to treat several pulmonary and urinary infections. The commercial form of gentamicin is a mixture of five compounds with minor differences in the methylation of one of their aminosugars. In the case of two compounds, gentamicin C2 and C2a, the only difference is the stereochemistry of the methyl group attached to C-6'. GenB2 is the enzyme responsible for this epimerization and is one of the four PLP-dependent enzymes encoded by the gentamicin biosynthetic gene cluster. Herein, we have determined the structure of GenB2 in its holo form in complex with PMP and also in the ternary complex with gentamicin X2 and G418, two substrate analogues. Based on the structural analysis, we were able to identify the structural basis for the catalytic mechanism of this enzyme, which was also studied by site-directed mutagenesis. Unprecedently, GenB2 is a PLP-dependent enzyme from fold I, which is able to catalyze an epimerization but with a mechanism distinct from that of fold III PLP-dependent epimerases using a cysteine residue near the N-terminus. The substitution of this cysteine residue for serine or alanine completely abolished the epimerase function of the enzyme, confirming its involvement. This study not only contributes to the understanding of the enzymology of gentamicin biosynthesis but also provides valuable details for exploring the enzymatic production of new aminoglycoside derivatives.
RESUMO
Chemical examination of an actinomycete strain Streptomyces malaysiensis DSM 4137 derived from a soil sample derived isolate Streptomyces sp. DSM 3816, yielded a new C2-asymmetric elaiophylin derivative efomycine U (1) and a known analogue halichoblelide D (2). These structures were unambiguously elucidated on the basis of extensive NMR spectroscopic and mass spectrometric analyses. All compounds isolated were subjected to antimicrobial, cytotoxic and immnosuppressive activities evaluation.
RESUMO
Modular polyketide synthase (PKS) is an ingenious core machine that catalyzes abundant polyketides in nature. Exploring interactions among modules in PKS is very important for understanding the overall biosynthetic process and for engineering PKS assembly-lines. Here, we show that intermodular recognition between the enoylreductase domain ER1/2 inside module 1/2 and the ketosynthase domain KS3 inside module 3 is required for the cross-module enoylreduction in azalomycin F (AZL) biosynthesis. We also show that KS4 of module 4 acts as a gatekeeper facilitating cross-module enoylreduction. Additionally, evidence is provided that module 3 and module 6 in the AZL PKS are evolutionarily homologous, which makes evolution-oriented PKS engineering possible. These results reveal intermodular recognition, furthering understanding of the mechanism of the PKS assembly-line, thus providing different insights into PKS engineering. This also reveals that gene duplication/conversion and subsequent combinations may be a neofunctionalization process in modular PKS assembly-lines, hence providing a different case for supporting the investigation of modular PKS evolution.
Assuntos
Policetídeo Sintases , Policetídeos , Policetídeo Sintases/genética , MacrolídeosRESUMO
A key step in the biosynthesis of numerous polyketides is the stereospecific formation of a spiroacetal (spiroketal). We report here that spiroacetal formation in the biosynthesis of the macrocyclic polyketides ossamycin and oligomycin involves catalysis by a novel spiroacetal cyclase. OssO from the ossamycin biosynthetic gene cluster (BGC) is homologous to OlmO, the product of an unannotated gene from the oligomycin BGC. The deletion of olmO abolished oligomycin production and led to the isolation of oligomycin-like metabolites lacking the spiroacetal structure. Purified OlmO catalyzed complete conversion of the major metabolite into oligomycin C. Crystal structures of OssO and OlmO reveal an unusual 10-strand ß-barrel. Three conserved polar residues are clustered together in the ß-barrel cavity, and site-specific mutation of any of these residues either abolished or substantially diminished OlmO activity, supporting a role for general acid/general base catalysis in spiroacetal formation.
Assuntos
Policetídeos , Antibacterianos , Catálise , Família Multigênica , Oligomicinas , Policetídeos/química , Metabolismo SecundárioRESUMO
Genomics-inspired isolation led to the identification of two new natural congeneric C2-asymmetric macrodiolide immunosuppressants, named efophylins A (1) and B (2), from Streptomyces malaysiensis DSM 4137. Their structures were elucidated by spectroscopic and computational methods and were in agreement with biosynthetic predictions from the efophylin gene cluster. Compound 2 exhibited potent immunosuppressive activity and demonstrated to inhibit the activation of the NFAT and block NFAT dephosphorylation in vitro. The immunosuppressive activity of compound 2 is possibly at least in part via the CaN/NFAT signaling pathway.
Assuntos
Produtos Biológicos/farmacologia , Imunossupressores/farmacologia , Linfócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Streptomyces/química , Animais , Produtos Biológicos/isolamento & purificação , Proliferação de Células , Feminino , Imunossupressores/isolamento & purificação , Teste de Cultura Mista de Linfócitos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Família Multigênica , Metabolismo Secundário , Baço/citologiaRESUMO
Adenylate-forming enzymes (AFEs) are a mechanistic superfamily of proteins that are involved in many cellular roles. In the biosynthesis of benzoxazole antibiotics, an AFE has been reported to play a key role in the condensation of cyclic molecules. In the biosynthetic gene cluster for the benzoxazole AJI9561, AjiA1 catalyzes the condensation of two 3-hydroxyanthranilic acid (3-HAA) molecules using ATP as a co-substrate. Here, the enzymatic activity of AjiA1 is reported together with a structural analysis of its apo form. The structure of AjiA1 was solved at 2.0â Å resolution and shows a conserved fold with other AFE family members. AjiA1 exhibits activity in the presence of 3-HAA (Km = 77.86 ± 28.36, kcat = 0.04 ± 0.004) and also with the alternative substrate 3-hydroxybenzoic acid (3-HBA; Km = 22.12 ± 31.35, kcat = 0.08 ± 0.005). The structure of AjiA1 in the apo form also reveals crucial conformational changes that occur during the catalytic cycle of this enzyme which have not been described for any other AFE member. Consequently, the results shown here provide insights into this protein family and a new subgroup is proposed for enzymes that are involved in benzoxazole-ring formation.
Assuntos
Modelos Moleculares , Conformação Proteica , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X/métodos , Especificidade por SubstratoRESUMO
The colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end-product, has become a cornerstone of knowledge-based genome mining. Herein, we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internal enoylreductase domain, previously identified as acting only in the second of two chain extension cycles on an initial iterative module, is shown to also catalyze enoylreduction in trans within the next module. The mechanism for this rare deviation from colinearity appears to involve direct cross-modular interaction of the reductase with the longer acyl chain, rather than back transfer of the substrate into the iterative module, suggesting an additional and surprising plasticity in natural PKS assembly-line catalysis.
Assuntos
Macrolídeos/metabolismo , Policetídeo Sintases/metabolismo , Biocatálise , Macrolídeos/química , Conformação Molecular , Oxirredução , Policetídeo Sintases/químicaRESUMO
The type I polyketide SF2487/A80577 (herein referred to as tetromadurin) is a polyether tetronate ionophore antibiotic produced by the terrestrial Gram-positive bacterium Actinomadura verrucosospora. Tetromadurin is closely related to the polyether tetronates tetronasin (M139603) and tetronomycin, all of which are characterised by containing a tetronate, cyclohexane, tetrahydropyran, and at least one tetrahydrofuran ring. We have sequenced the genome of Actinomadura verrucosospora to identify the biosynthetic gene cluster responsible for tetromadurin biosynthesis (the mad gene cluster). Based on bioinformatic analysis of the 32 genes present within the cluster a plausible biosynthetic pathway for tetromadurin biosynthesis is proposed. Functional confirmation of the mad gene cluster is obtained by performing in-frame deletions in each of the genes mad10 and mad31, which encode putative cyclase enzymes responsible for cyclohexane and tetrahydropyran formation, respectively. Furthermore, the A. verrucosospora Δmad10 mutant produces a novel tetromadurin metabolite that according to mass spectrometry analysis is analogous to the recently characterised partially cyclised tetronasin intermediate lacking its cyclohexane and tetrahydropyran rings. Our results therefore elucidate the biosynthetic machinery of tetromadurin biosynthesis and lend support for a conserved mechanism of cyclohexane and tetrahydropyran biosynthesis across polyether tetronates.
Assuntos
Macrolídeos/química , Policetídeo Sintases/genética , Policetídeos/metabolismo , Actinobacteria/enzimologia , Actinobacteria/metabolismo , Actinomadura , Sequência de Aminoácidos/genética , Antibacterianos/química , Sequência de Bases/genética , Vias Biossintéticas , Clonagem Molecular , Éteres/metabolismo , Furanos/metabolismo , Família Multigênica/genética , Alinhamento de SequênciaRESUMO
Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of Streptomyces conglobatus, here we discovered four new neoantimycin analogs, unantimycins B-E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety. Unantimycins B-E exhibited levels of anticancer activities similar to those of the chemotherapeutic drug cisplatin in human lung cancer, colorectal cancer, and melanoma cells. Notably, they mostly displayed no significant toxicity toward noncancerous cells, unlike the serious toxicities generally reported for antimycin-type natural products. Using site-directed mutagenesis and heterologous expression, we found that unantimycin productions are correlated with the activity of a chorismatase homolog, the nat-hyg5 gene, from a type I PKS gene cluster. Biochemical analysis confirmed that the catalytic activity of Nat-hyg5 generates 3-HBA from chorismate. Finally, we achieved selective production of unantimycins B and C by engineering a chassis host. On the basis of these findings, we propose that unantimycin biosynthesis is directed by the neoantimycin-producing NRPS-PKS complex and initiated with the starter unit of 3-HBA. The elucidation of the biosynthetic unantimycin pathway reported here paves the way to improve the yield of these compounds for evaluation in oncotherapeutic applications.
Assuntos
Antibióticos Antineoplásicos/biossíntese , Proteínas de Bactérias/metabolismo , Depsipeptídeos/biossíntese , Hidroxibenzoatos/química , Policetídeo Sintases/metabolismo , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular , Depsipeptídeos/química , Depsipeptídeos/toxicidade , Humanos , Compostos Orgânicos/química , Compostos Orgânicos/metabolismo , Compostos Orgânicos/toxicidade , Streptomyces/enzimologia , Streptomyces/metabolismoRESUMO
Whole genome analysis of Streptomyces sp. KO-7888 has revealed various pathway-specific transcriptional regulatory genes associated with silent biosynthetic gene clusters. A Streptomyces antibiotic regulatory protein gene, speR, located adjacent to a novel nonribosomal peptide synthetase (NRPS) gene cluster, was overexpressed in the wild-type strain. The resulting recombinant strain of Streptomyces sp. KO-7888 produced two new lipopeptides, sarpeptins A and B. Their structures were elucidated by high-resolution electrospray ionization mass spectrometry, NMR analysis, and the advanced Marfey's method. The distinct modular sections of the corresponding NRPS biosynthetic gene cluster were characterized, and the assembly line for production of the lipopeptide chain was proposed.
Assuntos
Lipopeptídeos/isolamento & purificação , Peptídeo Sintases/metabolismo , Streptomyces/metabolismo , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Fungos/efeitos dos fármacos , Genes Bacterianos , Genes Reguladores , Lipopeptídeos/biossíntese , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Testes de Sensibilidade Microbiana , Conformação Proteica , Streptomyces/genéticaRESUMO
Ossamycin from Streptomyces hygroscopicus var. ossamyceticus is an antifungal and cytotoxic polyketide and a potent inhibitor of the mitochondrial ATPase. Analysis of a near-complete genome sequence of the ossamycin producer has allowed the identification of the 127-kbp ossamycin biosynthetic gene cluster. The presence in the cluster of a specific crotonyl-CoA carboxylase/reductase homologue suggests that the 5-methylhexanoate extension unit used in construction of the macrocyclic core is incorporated intact from the unusual precursor isobutyrylmalonyl-CoA. Surprisingly, the modular polyketide synthase uses only 14 extension modules to accomplish 15 cycles of polyketide chain extension, a rare example of programmed iteration on a modular polyketide synthase. Specific deletion of genes encoding cytochrome P450 enzymes has given insight into the late-stage tailoring of the ossamycin macrocycle required for the attachment of the unusual 2,3,4,6-deoxyaminohexose sugar l-ossamine to C-8 of the ossamycin macrocycle. The ossamycin cluster also encodes a putative spirocyclase enzyme, OssO, which may play a role in establishing the characteristic spiroketal moiety of the natural product.
Assuntos
Vias Biossintéticas , Macrolídeos/química , Macrolídeos/metabolismo , Compostos de Espiro/química , Vias Biossintéticas/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Genes Bacterianos , Família Multigênica , Policetídeo Sintases/genética , Streptomyces/genéticaRESUMO
Malayamycin A is an unusual bicyclic C-nucleoside, with interesting antiviral, antifungal, and anticancer bioactivity. We report here the discovery and characterization of the biosynthetic pathway to malayamycin by using genome mining of near-identical clusters both from the known producer Streptomyces malaysiensis and from Streptomyces chromofuscus. The key precursor 5'-pseudouridine monophosphate (5'-Ψ-MP) is supplied chiefly through the action of MalD, a TruD-like pseudouridine synthase. In vitro assays showed that MalO is an enoylpyruvyltransferase acting almost exclusively on 5'-Ψ-MP rather than 5'-UMP, while in contrast the counterpart enzyme NikO in the nikkomycin pathway readily accepts either substrate. As a result, deletion of malD in S. chromofuscus coupled with introduction of the gene for NikO led to production of non-natural N-malayamycin, as well as malayamycin A. Conversely, cloning malO into the nikkomycin producer Streptomyces tendae in place of nikO diverted biosynthesis toward C-nucleoside formation.
Assuntos
Antifúngicos/metabolismo , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Nucleosídeos/metabolismo , Streptomyces/metabolismo , Aminoglicosídeos/genética , Aminoglicosídeos/metabolismo , Proteínas de Bactérias/genética , Genoma Bacteriano , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Família Multigênica , Nucleosídeos/genética , Streptomyces/genéticaRESUMO
Enzymes often convert both physiological and non-physiological substrates with high stereoselectivity; yet, for some enzymes, opposite product chirality is observed. A possible explanation is the existence of hidden specificities becoming apparent when non-physiological substrates confer different substrate-enzyme interactions than the physiological substrate. To test this hypothesis, a series of α-methylated ß-keto esters were converted with Tyl-KR1, a ketoreductase from polyketide synthesis in Streptomyces fradiae. The conversions of six substrates with different physicochemical properties exhibited enantioselectivities ranging from 84 % ee for R,R to 84 % ee for S,S, yet high and uniform diastereoselectivity (anti, d.r.>9:1). The exchange of a single atom, namely an oxygen ester instead of a thioester, led to almost complete loss of enantioselectivity (<5 % ee). An additional S,S-selective binding mode as a hidden specificity in Tyl-KR1 has been identified through molecular modeling and site-directed mutagenesis.
Assuntos
Oxirredutases do Álcool/química , Proteínas de Bactérias/química , Cetonas/química , Oxirredutases do Álcool/genética , Álcoois/síntese química , Álcoois/química , Proteínas de Bactérias/genética , Biocatálise , Mutação , Oxirredução , Estereoisomerismo , Streptomyces/enzimologia , Especificidade por SubstratoRESUMO
Neoantimycins (NATs) are members of antimycin-types of depsipeptides with outstanding anticancer activities. We isolated NAT-A (1) and -F (2) from the fermentation extract of Streptomyces conglobatus. The NAT biosynthetic gene cluster ( nat BGC) was identified by genome sequencing and bioinformatics analysis. nat BGC includes two nonribosomal peptide synthetase (NRPS) and one polyketide synthase (PKS) gene, and a gene cassette (10 genes), of which the encoded enzymes share high homology to the ones responsible for 3-formamidosalicylate (3-FAS) biosynthesis in the antimycin biosynthetic pathway. Heterologous expression of the partial nat BGC without the 3-FAS gene cassette in the antimycin producer, Streptomyces albus J1074, results in the production of 1 and 2, suggesting that the nat BGC indeed directs NATs biosynthesis. Targeted in-frame deletion of the reductase gene ( natE) abolished the production of 1 and 2 but accumulated two NAT derivatives, the known NAT-H (3) and a new NAT-I (4). Biochemical verification demonstrated that the recombinant NatE indeed catalyzes an NADPH-dependent reaction of 3 or 4 to 1 or 2, respectively. Compound 3 presented significantly stronger activities against eight cancer cell lines than the ones using cisplatin, the clinical chemotherapy medicine. In particular, 3 displayed 559- and 57-fold higher activity toward human melanoma and cervix epidermoid carcinoma cells, respectively, compared with cisplatin. The new derivative, 4, was 1.5- to 10.9-fold more active than cisplatin toward five cancer cell lines. The evaluation of NATs biosynthesis depicted here will pave the way to generate new NAT derivatives through rational pathway engineering.
Assuntos
Antineoplásicos/metabolismo , Vias Biossintéticas , Depsipeptídeos/metabolismo , Oxirredutases/metabolismo , Streptomyces/enzimologia , Streptomyces/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Depsipeptídeos/genética , Depsipeptídeos/farmacologia , Genes Bacterianos , Humanos , Família Multigênica , NADP/metabolismo , Neoplasias/tratamento farmacológico , Compostos Orgânicos/metabolismo , Compostos Orgânicos/farmacologia , Oxirredutases/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Streptomyces/genéticaRESUMO
Gentamicin C complex from Micromonospora echinospora remains a globally important antibiotic, and there is revived interest in the semisynthesis of analogs that might show improved therapeutic properties. The complex consists of five components differing in their methylation pattern at one or more sites in the molecule. We show here, using specific gene deletion and chemical complementation, that the gentamicin pathway up to the branch point is defined by the selectivity of the methyltransferases GenN, GenD1, and GenK. Unexpectedly, they comprise a methylation network in which early intermediates are ectopically modified. Using whole-genome sequence, we have also discovered the terminal 6'-N-methyltransfer required to produce gentamicin C2b from C1a or gentamicin C1 from C2, an example of an essential biosynthetic enzyme being located not in the biosynthetic gene cluster but far removed on the chromosome. These findings fully account for the methylation pattern in gentamicins and open the way to production of individual gentamicins by fermentation, as starting materials for semisynthesis.
Assuntos
Gentamicinas/biossíntese , Metiltransferases/metabolismo , Micromonospora/enzimologia , Micromonospora/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Deleção de Genes , Gentamicinas/metabolismo , Metilação , Metiltransferases/genética , Micromonospora/metabolismo , Família Multigênica , Mutação , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
Clethramycin from Streptomyces malaysiensis DSM4137, and mediomycins (produced together with clethramycin from Streptomyces mediocidicus), are near-identical giant linear polyenes apparently constructed from, respectively, a 4-guanidinobutanoate or 4-aminobutanoate starter unit and 27 polyketide extender units, and bearing a specific O-sulfonate modification at the C-29 hydroxy group. We show here that mediomycins are actually biosynthesised not by use of a different starter unit but by direct late-stage deamidination of (desulfo)clethramycin. A gene (slf) encoding a candidate sulfotransferase has been located in both gene clusters. Deletion of this gene in DSM4137 led to accumulation of desulfoclethramycin only, instead of a mixture of desulfoclethramycin and clethramycin. The mediomycin gene cluster does not encode an amidinohydrolase, but when three candidate amidinohydrolase genes from elsewhere in the S. mediocidicus genome were individually expressed in Escherichia coli and assayed, only one of them (medi4948), located 670 kbp away from the mediomycin gene cluster on the chromosome, catalysed the removal of the amidino group from desulfoclethramycin. Subsequent cloning of medi4948 into DSM4137 caused mediomycins A and B to accumulate at the expense of clethramycin and desulfoclethramycin, respectively, a rare case where an essential biosynthetic gene is not co-located with other pathway genes. Clearly, both desulfoclethramycin and clethramycin are substrates for this amidinohydrolase. Also, purified recombinant sulfotransferase from DSM4137, in the presence of 3'-phosphoadenosine-5'-phosphosulfate as donor, efficiently converted mediomycin B to mediomycin A in vitro. Thus, in the final steps of mediomycin A biosynthesis deamidination and sulfotransfer can take place in either order.
RESUMO
Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.
Assuntos
Vias Biossintéticas/genética , Evolução Molecular , Variação Genética , Família Multigênica , Bioengenharia , Policetídeo Sintases/genética , Sirolimo/química , Sirolimo/metabolismoRESUMO
Gentamicins are heavily methylated, clinically valuable pseudotrisaccharide antibiotics produced by Micromonospora echinospora. GenN has been characterized as an S-adenosyl-l-methionine-dependent methyltransferase with low sequence similarity to other enzymes. It is responsible for the 3â³-N-methylation of 3â³-dehydro-3â³-amino-gentamicin A2, an essential modification of ring III in the biosynthetic pathway to the gentamicin C complex. Purified recombinant GenN also efficiently catalyzes 3â³-N-methylation of related aminoglycosides kanamycin B and tobramycin, which both contain an additional hydroxymethyl group at the C5â³ position in ring III. We have obtained eight cocrystal structures of GenN, at a resolution of 2.2 Å or better, including the binary complex of GenN and S-adenosyl-l-homocysteine (SAH) and the ternary complexes of GenN, SAH, and several aminoglycosides. The GenN structure reveals several features not observed in any other N-methyltransferase that fit it for its role in gentamicin biosynthesis. These include a novel N-terminal domain that might be involved in protein:protein interaction with upstream enzymes of the gentamicin X2 biosynthesis and two long loops that are involved in aminoglycoside substrate recognition. In addition, the analysis of structures of GenN in complex with different ligands, supported by the results of active site mutagenesis, has allowed us to propose a catalytic mechanism and has revealed the structural basis for the surprising ability of native GenN to act on these alternative substrates.
Assuntos
Aminoglicosídeos/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Gentamicinas/metabolismo , Metiltransferases/metabolismo , Micromonospora/enzimologia , Proteínas de Bactérias/química , Cristalografia por Raios X , Canamicina/análogos & derivados , Canamicina/metabolismo , Metiltransferases/química , Micromonospora/química , Micromonospora/metabolismo , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato , Tobramicina/metabolismoRESUMO
Detailed analysis of the modular Typeâ I polyketide synthase (PKS) involved in the biosynthesis of the marginolactone azalomycinâ F in mangrove Streptomyces sp. 211726 has shown that only nineteen extension modules are required to accomplish twenty cycles of polyketide chain elongation. Analysis of the products of a PKS mutant specifically inactivated in the dehydratase domain of extension-module 1 showed that this module catalyzes two successive elongations with different outcomes. Strikingly, the enoylreductase domain of this module can apparently be "toggled" off and on : it functions in only the second of these two cycles. This novel mechanism expands our understanding of PKS assembly-line catalysis and may explain examples of apparent non-colinearity in other modular PKS systems.
Assuntos
Macrolídeos/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Macrolídeos/química , Conformação Molecular , Mutação , Oxirredutases/genética , Policetídeo Sintases/genéticaRESUMO
Genome sequencing of Streptomyces malaysiensis DSM 4137 revealed the presence of four terpene cyclase genes, one of which was characterised as (+)-isoafricanol synthase. Its cyclisation mechanism was extensively studied using isotopically labelled precursors. Several enzymes with high homology that likely also function as (+)-isoafricanol synthases are encoded in a number of other genome sequenced streptomycetes.