RESUMO
A novel actinomycete, designated as TPMA0078T, was isolated from a soil sample collected in Shinjuku, Tokyo, Japan. 16S rRNA gene sequence analysis indicated that strain TPMA0078T belongs to the genus Actinoplanes and is closely related to Actinoplanes regularis IFO 12514T (99.86% 16S rRNA gene sequence similarity). The spores of strain TPMA0078T were motile, and the sporangia were cylindrical. The diamino acids in the cell wall peptidoglycan of strain TPMA0078T were meso-diaminopimelic acid and 3OH-meso-diaminopimelic acid. Whole-cell sugars were glucose and mannose, with galactose as a minor component. The major cellular fatty acids identified were iso-C15:0, iso-C16:0, and anteiso-C17:0. The predominant menaquinone was MK-9(H4), and the principal polar lipid was phosphatidylethanolamine. These chemotaxonomic properties of strain TPMA0078T were consistent with those of Actinoplanes. Meanwhile, digital DNA-DNA hybridization and average nucleotide identity values showed low relatedness between strain TPMA0078T and A. regularis NBRC 12514T. Furthermore, several phenotypic properties of strain TPMA0078T distinguished it from those of closely related species. Based on its genotypic and phenotypic characteristics, strain TPMA0078T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes kirromycinicus sp. nov. is proposed. The type strain is TPMA0078T (=NBRC 116422T = TBRC 18262T).
RESUMO
A triple mutant (strain KA57) of Streptomyces rochei 7434AN4 produces an azoxy-alkene compound, KA57A, which was not detected in a parent strain or other single and double mutants. This strain accumulated several additional minor components, whose structures were elucidated. HPLC analysis of strain KA57 indicated the presence of two UV active components (KA57D1 and KA57D2) as minor components. They exhibited a maximum UV absorbance at 218 nm, whereas a UV absorbance of azoxy-alkene KA57A was detected at 236 nm, suggesting that both KA57D1 and KA57D2 contain a different chromophore from KA57A. KA57D1 has a molecular formula of C12H22N2O2, and NMR analysis revealed KA57D1 is a novel hydrazide-alkene compound, (Z)-N-acetyl-N'-(hex-1-en-1-yl)isobutylhydrazide. Labeling studies indicated that nitrogen Nß of KA57D1 is derived from l-glutamic acid, and the isobutylamide unit (C-1 to C-3, 2-Me, and Nα) originates from valine. KA57D2 has a molecular formula of C13H24N2O2, and its structure was determined to be (Z)-N-acetyl-N'-(hex-1-en-1-yl)-2-methylbutanehydrazide, in which a 2-methylbutanamide unit was shown to originate from isoleucine. Different biogenesis of the Nα atom (l-serine for KA57A, l-valine for KA57D1, and l-isoleucine for KA57D2) indicates the relaxed substrate recognition for nitrogen-nitrogen bond formation in the biosyntheses of KA57A, KA57D1, and KA57D2.
Assuntos
Aminoácidos , Streptomyces , Aminoácidos/metabolismo , Alcenos , Streptomyces/genética , Streptomyces/metabolismoRESUMO
A new maniwamycin analogue, maniwamycin G, was isolated from Streptomyces sp. TOHO-M025 as a major product. Maniwamycin G has a molecular formula of C12H22N2O4, and its extensive NMR analysis revealed that maniwamycin G contains a methoxycarbonyl group instead of an amide as found in maniwamycin F. Its C-2 and C-3 configurations were determined to be (2R, 3R) by circular dichroism spectrum and a modified Mosher method, respectively. The biosynthetic origin of maniwamycin G was investigated using isotope-labeled compounds. The carbon source of maniwamycin G is four acetate units (C-1', C-2'; C-3', C-4'; C-5', C-6'; and C-4, C-5) and l-serine (C-1 to C-3). The nitrogen atom attached at C-2 (Nα) originates from serine, whereas the nitrogen atom of a hexen-1-yl amine unit (Nß) is derived from glutamic acid. The quorum-sensing inhibitory activity of maniwamycin G was 2-fold lower than that of maniwamycin F.
Assuntos
Streptomyces , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Nitrogênio , Percepção de Quorum , Streptomyces/químicaRESUMO
MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain Micromonospora griseorubida A11725, very low-level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite. Thus, mycG was subject to random mutagenesis and screening was conducted in Escherichia coli whole-cell assays. This enabled efficient identification of amino acid residues involved in reaction profile alterations, which included MycG R111Q/V358L, W44R, and V135G/E355K with enhanced monohydroxylation to accumulate mycinamicin V. The MycG V135G/E355K mutant generated 40-fold higher levels of mycinamicin V compared to wild-type M. griseorubida A11725. In addition, the E355K mutation showed improved ability to catalyze sequential hydroxylation and epoxidation with minimal mono-epoxidation product mycinamicin I compared to the wild-type enzyme. These approaches demonstrate the ability to selectively coordinate the catalytic activity of multifunctional P450s and efficiently produce the desired compounds.
Assuntos
Sistema Enzimático do Citocromo P-450 , Macrolídeos , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxilação , Oxirredução , Biossíntese PeptídicaRESUMO
Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens.
Assuntos
Macrolídeos/química , Micromonospora/química , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Eritromicina/química , Humanos , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidadeRESUMO
A novel actinomycete, designated NUM-2625T, was isolated as an endophytic bacterium in aerial parts of Comarum salesowianum, an endemic species in the Altai, Himalaya mountain chain area, collected from Khasagt Khairkhan Mountain in Mongolia. The 16S rRNA gene sequence of strain NUM-2625T showed the highest similarity to Actinocatenispora thailandica TT2-10T (99.4â%), Actinocatenispora sera KV-744T (99.3â%), and Actinocatenispora rupis CS5-AC17T (97.7â%). Chemotaxonomic properties of strain NUM-2625T were essentially consistent with those of the genus Actinocatenispora, such as the presence of meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan, MK-9(H4) and MK-9(H6) as the major menaquinones, and iso-C16â:â0, iso-C15â:â0, iso-C14â:â0 3-OH, and anteiso-C17â:â0 as the major fatty acids. Meanwhile, digital DNA-DNA hybridization and average nucleotide identity values revealed a low relatedness between strain NUM-2625T and the other type strains of the genus Actinocatenispora. In addition, strain NUM-2625T exhibited several phenotypic properties that could be used to distinguish it from its closest relatives. Based on the results of polyphasic analyses, strain NUM-2625T represents a novel species in the genus Actinocatenispora, for which the name Actinocatenispora comari sp. nov. is proposed. The type strain is NUM-2625T (=NBRC 114660T=TBRC 13496T).
Assuntos
Micromonosporaceae/classificação , Filogenia , Componentes Aéreos da Planta/microbiologia , Rosácea/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácido Diaminopimélico/química , Endófitos/classificação , Endófitos/isolamento & purificação , Ácidos Graxos/química , Micromonosporaceae/isolamento & purificação , Mongólia , Hibridização de Ácido Nucleico , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
Cytochrome P450 enzymes (P450s) are one of the major factors responsible for the diversity of metabolites produced through many biosynthetic and biodegradative processes in actinomycetes. P450s typically catalyze a single oxidative modification; however, several P450s have been identified with the unique ability to iteratively oxidize the same-site of the substrate. These P450s are capable of forming diverse compounds that affect biological processes, including alcohols, ketones, aldehydes, and carboxylic acids. Although further structural and functional studies are needed to elucidate the mechanisms that allow multistep oxidative modification, recent studies have revealed the enzymatic properties and reaction mechanisms of these P450s. This mini-review covers the current knowledge of P450s that catalyze the multistep oxidation reactions and contribute to the production of a wide variety of metabolites by selected actinomycete strains, along with insights into their application and utility. Understanding the characteristics of these remarkable enzymes will facilitate their utilization in biotechnological applications to create biologically active and other high-value compounds. KEY POINTS: ⢠The multistep oxidation by P450s plays a key role in the diversity of metabolites. ⢠The mechanisms that enable P450s to catalyze iterative oxidation remains unknown. ⢠The effective use of P450s that iteratively oxidize the same-site is discussed.
Assuntos
Actinobacteria , Actinobacteria/metabolismo , Biotecnologia , Catálise , Sistema Enzimático do Citocromo P-450/metabolismo , OxirreduçãoRESUMO
MycG is a cytochrome P450 that performs two sequential oxidation reactions on the 16-membered ring macrolide M-IV. The enzyme evolved to oxidize M-IV preferentially over M-III and M-VI, which differ only by the presence of methoxy vs free hydroxyl groups on one of the macrolide sugar moieties. We utilized a two-pronged computational approach to study both the chemoselective reactivity and substrate specificity of MycG. Density functional theory computations determined that epoxidation of the substrate hampers its ability to undergo C-H abstraction, primarily due to a loss of hyperconjugation in the transition state. Metadynamics and molecular dynamics simulations revealed a hydrophobic sugar-binding pocket that is responsible for substrate recognition/specificity and was not apparent in crystal structures of the enzyme/substrate complex. Computational results also led to the identification of other interactions between the enzyme and its substrates that had not previously been observed in the cocrystal structures. Site-directed mutagenesis was then employed to test the effects of mutations hypothesized to broaden the substrate scope and alter the product profile of MycG. The results of these experiments validated this complementary effort to engineer MycG variants with improved catalytic activity toward earlier stage mycinamicin substrates.
Assuntos
Antibacterianos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Macrolídeos/metabolismo , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Antibacterianos/química , Sistema Enzimático do Citocromo P-450/química , Macrolídeos/química , Conformação Molecular , OxirreduçãoRESUMO
The cytochrome P450 monooxygenase RosC catalyzes the three-step oxidation reactions, which leads to the formation of a hydroxy, formyl, and carboxy group at C-20 during rosamicin biosynthesis in Micromonospora rosaria IFO13697. To determine if amino acid substitutions in RosC could allow for the control of the multistep oxidation reactions, we screened RosC random mutants. The RosC mutant RM30, with five amino acid substitutions (P107S, L176Q, S254N, V277A, and I319N), catalyzed only the first step of the oxidation reaction. Whole-cell assays using Escherichia coli cells expressing RosC mutants with single and double amino acid substitutions derived from RM30 indicated that P107S/L176Q, P107S/V277A, P107S/I319N, L176Q/V277A, L176Q/I319N, and S254N/V277A significantly reduced the catalytic activity of the second reaction, which is alcohol oxidation. Of the previously mentioned mutants, double mutants containing L176Q, which was presumed to occur in the FG loop region, lost the total catalytic activity of the third reaction (aldehyde oxidation). Additionally, an engineered M. rosaria strain with rosC disruption, which introduced the gene encoding the RosC mutants P107S/L176Q and P107S/V277A preferentially produced 20-dihydrorosamicin, which is formed after the first oxidation reaction of RosC.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Leucomicinas/biossíntese , Micromonospora/enzimologia , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Escherichia coli/genética , Mutação , OxirreduçãoRESUMO
Quorum sensing (QS) is a microbial signaling system that regulates the expression of many virulence genes. Herein, we studied five compounds-No. 1: (E)-2-methyl-3- (4-nitro-phenyl)-acrylaldehyde; No. 29-2: pimprinine [5-(1H-indol-3-yl)-2-methyloxazole]; No. 48: (2E,4E)-2-methyl-5-phenyl-2,4-pentadienoic acid; No. 74: (3E,5E)-5-methyl-6-(4-nitrophenyl)-hexa-3,5-dien-2-ol; and No. 130: methyphenazine-1-carboxylate-derived from an actinomycete metabolite library. These compounds were confirmed to be QS inhibitors that reduced violacein production in Chromobacterium violaceum CV026. Additionally, compounds No. 1, No. 74, and No. 130 significantly reduced fluorescent pigment production in Pseudomonas aeruginosa ATCC 27853.
Assuntos
Actinobacteria/metabolismo , Chromobacterium/metabolismo , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum , Indóis/metabolismo , Pigmentos Biológicos/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimentoRESUMO
The discovery that the actinobacterium Micromonospora inhabits nitrogen-fixing nodules raised questions as to its potential ecological role. The capacity of two Micromonospora strains to infect legumes other than their original host, Lupinus angustifolius, was investigated using Medicago and Trifolium as test plants. Compatible rhizobial strains were used for coinoculation of the plants because Micromonospora itself does not induce nodulation. Over 50% of nodules from each legume housed Micromonospora, and using 16S rRNA gene sequence identification, we verified that the reisolated strains corresponded to the microorganisms inoculated. Entry of the bacteria and colonization of the plant hosts were monitored using a GFP-tagged Lupac 08 mutant together with rhizobia, and by using immunogold labeling. Strain Lupac 08 was localized in plant tissues, confirming its capacity to enter and colonize all hosts. Based on studying three different plants, our results support a non-specific relationship between Micromonospora and legumes. Micromonospora Lupac 08, originally isolated from Lupinus re-enters root tissue, but only when coinoculated with the corresponding rhizobia. The ability of Micromonospora to infect and colonize different legume species and function as a potential plant-growth promoting bacterium is relevant because this microbe enhances the symbiosis without interfering with the host and its nodulating and nitrogen-fixing microbes.
Assuntos
Medicago/microbiologia , Interações Microbianas , Micromonospora/crescimento & desenvolvimento , Rhizobiaceae/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/microbiologia , Trifolium/microbiologia , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Lupinus/microbiologia , Micromonospora/classificação , Micromonospora/genética , Micromonospora/isolamento & purificação , Desenvolvimento Vegetal , Doenças das Plantas , RNA Ribossômico 16S/genética , Rhizobiaceae/classificação , Rhizobiaceae/genética , Rhizobiaceae/isolamento & purificação , Análise de Sequência de DNARESUMO
The cytochrome P450 enzyme RosC catalyzes a two-step, hydroxylation and alcohol oxidation, oxidation reaction to form the C-20 formyl group in the biosynthesis of a 16-membered macrolide antibiotic rosamicin produced by Micromonospora rosaria IFO13697. RosC is presumed to be involved in the formation of 20-carboxyrosamicin because it has been isolated from the culture broth of M. rosaria. Here, we confirmed that RosC has catalytic activity, with E. coli expressing RosC converting rosamicin into 20-carboxyrosamicin. Therefore, it was revealed that RosC is a multifunctional P450 that catalyzes a three-step oxidation reaction that leads to the formation of the hydroxyl group, formyl group and carboxyl group at C-20 on the macrolactone ring in the rosamicin biosynthetic pathway. Moreover, the cytochrome P450 enzyme TylI, which is involved in formation of the formyl group of a 16-membered macrolide antibiotic tylosin produced by Streptomyces fradiae ATCC 19609, also converted rosamicin into 20-carboxyrosamicin.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Leucomicinas/metabolismo , Macrolídeos/metabolismo , Antibacterianos/metabolismo , Biocatálise , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroxilação , Micromonospora/metabolismo , Oxirredução , Streptomyces/metabolismoRESUMO
Quorum sensing is an important microbial signaling system that controls the expression of many virulence genes. Maniwamycins C-F, new compounds and quorum-sensing inhibitors, were isolated from the culture broth of Streptomyces sp. TOHO-M025 using a silica gel column and preparative HPLC. The structures of maniwamycins were elucidated by spectroscopic analyses, including NMR. The compounds each have an azoxy moiety. All maniwamycins inhibited violacein synthesis, which is controlled by quorum sensing, in Chromobacterium violaceum CV026.
Assuntos
Antibacterianos/farmacologia , Compostos Azo/farmacologia , Chromobacterium/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Streptomyces/metabolismo , Compostos Azo/isolamento & purificação , Indóis/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates, show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homologue, active site residues were identified that correlate with the 3' or 4' specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.
Assuntos
Metiltransferases/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Metiltransferases/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Genetic engineering of post-polyketide synthase-tailoring genes can be used to generate new macrolide analogs through manipulation of the genes involved in their biosynthesis. Rosamicin, a 16-member macrolide antibiotic produced by Micromonospora rosaria IFO13697, contains a formyl group and an epoxide at C-20 and C-12/13 positions which are formed by the cytochrome P450 enzymes RosC and RosD, respectively. The D-mycinose biosynthesis genes in mycinamicin II biosynthesis gene cluster of Micomonospora guriseorubida A11725 were introduced into the rosC and rosD disruption mutants of M. rosaria IFO13697. The resulting engineered strains, M. rosaria TPMA0054 and TPMA0069, produced mycinosyl rosamicin derivatives, IZIV and IZV, respectively. IZIV was identified as a novel mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydrorosamicin.
Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Sistema Enzimático do Citocromo P-450/genética , Leucomicinas/biossíntese , Micromonospora/genética , Micromonospora/metabolismo , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/metabolismo , Engenharia Genética , Leucomicinas/química , Micromonospora/enzimologia , Estrutura Molecular , Mutação , Policetídeo Sintases/genéticaRESUMO
Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Domínio Catalítico , Macrolídeos/metabolismo , Modelos Moleculares , Oxirredução , Ligação ProteicaRESUMO
Much attention has been paid to cholestane-type steroidal glycosides because of their importance from the perspectives of both chemical diversity and significant biological activities. A phytochemical investigation of the rhizomes of Polygonatum odoratum (Liliaceae) resulted in the isolation of three novel cholestane-type steroidal glycosides (1-3) with unique Δ(14,16)-unsaturated D-ring structures as well as two novel spirostane-type steroidal saponins (4 and 5) and three known steroidal glycosides (6-8). Their structures were determined by various spectroscopic methods and chemical reactions. Steroidal saponin 7 showed significant antifungal activity against Candida albicans JCM1542 (MIC 3.1 µg/mL) and Aspergillus fumigatus JCM1738 (MIC 6.3 µg/mL).
Assuntos
Colestanos/química , Glicosídeos/química , Extratos Vegetais/química , Polygonatum/química , Saponinas/química , Espirostanos/química , Colestanos/isolamento & purificação , Glicosídeos/isolamento & purificação , Estrutura Molecular , Saponinas/isolamento & purificação , Espirostanos/isolamento & purificação , EstereoisomerismoRESUMO
The cytochrome P450 enzyme-encoding genes rosC and rosD were cloned from the rosamicin biosynthetic gene cluster of Micromonospora rosaria IFO13697. The functions of RosC and RosD were demonstrated by gene disruption and complementation with M. rosaria and bioconversion of rosamicin biosynthetic intermediates with Escherichia coli expressing RosC and RosD. It is proposed that M. rosaria IFO13697 has two pathway branches that lead from the first desosaminyl rosamicin intermediate, 20-deoxo-20-dihydro-12,13-deepoxyrosamicin, to rosamicin.
Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Leucomicinas/biossíntese , Micromonospora/enzimologia , Micromonospora/genética , Proteínas de Bactérias/genética , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Deleção de Genes , Teste de Complementação Genética , Família MultigênicaRESUMO
The majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthetic steps, leading to oxidative tailoring of the 16-membered ring macrolide antibiotic mycinamicin II in the actinomycete Micromonospora griseorubida. The ordering of steps to complete the biosynthetic process involves a complex substrate recognition pattern by the enzyme and interplay between three tailoring modifications as follows: glycosylation, methylation, and oxidation. To understand the catalytic properties of MycG, we structurally characterized the ligand-free enzyme and its complexes with three native metabolites. These include substrates mycinamicin IV and V and their biosynthetic precursor mycinamicin III, which carries the monomethoxy sugar javose instead of the dimethoxylated sugar mycinose. The two methoxy groups of mycinose serve as sensors that mediate initial recognition to discriminate between closely related substrates in the post-polyketide oxidative tailoring of mycinamicin metabolites. Because x-ray structures alone did not explain the mechanisms of macrolide hydroxylation and epoxidation, paramagnetic NMR relaxation measurements were conducted. Molecular modeling based on these data indicates that in solution substrate may penetrate the active site sufficiently to place the abstracted hydrogen atom of mycinamicin IV within 6 Å of the heme iron and ~4 Å of the oxygen of iron-ligated water.
Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Macrolídeos/metabolismo , Micromonospora/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Compostos de Epóxi/metabolismo , Glicosilação , Hidroxilação , Macrolídeos/química , Espectroscopia de Ressonância Magnética , Metilação , Micromonospora/genética , Micromonospora/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutação , Oxirredução , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Some polyketide-derived bioactive compounds contain sugars attached to the aglycone core, and these sugars often enhance or impart specific biological activity to the molecule. Mycinamicin II, a 16-member macrolide antibiotic produced by Micromonospora griseorubida A11725, contains a branched lactone and two different deoxyhexose sugars, D-desosamine and D-mycinose, at the C-5 and C-21 positions, respectively. We previously engineered an expression plasmid pSETmycinose containing the D-mycinose biosynthesis genes from M. griseorubida A11725. This plasmid was introduced into Micromonospora sp. FERM BP-1076 cells, which produce the 16-membered macrolide antibiotic izenamicin. The resulting engineered strain TPMA0041 produced 23-O-mycinosyl-20-deoxy-izenamicin B(1) and 22-O-mycinosyl-izenamicin B(2). 23-O-mycinosyl-20-deoxy-izenamicin B(1) has been produced by the engineered strain M. rosaria TPMA0001 containing pSETmycinose as 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (=IZI) in our recent study, and 22-O-mycinosyl-izenamicin B(2) has previously been synthesized as a macrolide antibiotic TMC-016 with strong antibacterial activity. The production of 22-O-mycinosyl-izenamicin B(2) (=TMC-016) was increased when propionate, a precursor of methylmalonyl-CoA, was added to the culture broth.