RESUMO
Geldanamycin and its derivatives, inhibitors of heat shock protein 90, are considered potent anticancer drugs, although their biosynthetic pathways have not yet been fully elucidated. The key step of conversion of 4,5-dihydrogeldanamycin to geldanamycin was expected to catalyze by a P450 monooxygenase, Gel16. The adequate bioconversions by cytochrome P450 mostly rely upon its interaction with redox partners. Several ferredoxin and ferredoxin reductases are available in the genome of certain organisms, but only a few suitable partners can operate in full efficiency. In this study, we have expressed cytochrome P450 gel16 in Escherichia coli and performed an in vitro assay using 4,5-dihydrogeldanamycin as a substrate. We demonstrated that the in silico method can be applicable for the efficient mining of convenient endogenous redox partners (9 ferredoxins and 6 ferredoxin reductases) against CYP Gel16 from Streptomyces hygroscopicus. The distances for ligand FDX4-FDR6 were found to be 9.384 Å. Similarly, the binding energy between Gel16-FDX4 and FDX4-FDR6 were -611.88 kcal/mol and -834.48 kcal/mol, respectively, suggesting the lowest distance and binding energy rather than other redox partners. These findings suggest that the best redox partners of Gel16 could be NADPH â FDR6 â FDX4 â Gel16.
Assuntos
Proteínas de Bactérias/metabolismo , Benzoquinonas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Transporte de Elétrons , Ferredoxinas/metabolismo , Lactamas Macrocíclicas/metabolismo , NADP/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Vias Biossintéticas , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Simulação de Acoplamento Molecular , Oxirredução , Ligação Proteica , Streptomyces/genética , Streptomyces/metabolismoRESUMO
To evaluate high-pressure processing combined with enzymatic treatment for extraction of Prunus mume by determining the optimum extraction conditions for the antimicrobial activity against oral bacteria. Highpressure enzymatic extraction was used to isolate biologically active components from P. mume. The effects of process variables such as enzyme type (Pectinex Ultra SP-L, Novozym 33095 and Viscozyme L), enzyme concentration, incubation time/temperature, pH, and ratio of enzymes antimicrobial activity against oral pathogens related to dental caries and periodontal diseases were determined by disk diffusion assay. The optimal conditions for enzymatic extraction from P. mume were pH 6.0, 45°C, 20 h, and 5 v/v% with Pectinex Ultra SP-L. The maximum antimicrobial activity of P. mume extract obtained using Novozym 33095 was at pH 7.0, 45°C, 20 h, and 5 v/v%. The Viscozyme L extract showed the maximum inhibitory effect at pH 6.0, 45°C, 20 h, and 5 v/v%. Use of combinations of enzymes did not result in significantly different antimicrobial activity (p < 0.05) compared with each enzyme alone. Minimum inhibitory concentration values were 3.125 to 12.50%. These results indicated that high-pressure enzymatic extraction yielded P. mume extract with antimicrobial activity which has the potential for improving oral environment.
Avaliar o processamento de alta pressão combinado com o tratamento enzimático para a extração de Prunus mume, determinando as condições ótimas de extração para a atividade antimicrobiana contra as bactérias orais. A extração enzimática de alta pressão foi utilizada para isolar os componentes biologicamente ativos de P. mume. Os efeitos das variáveis do processo como o tipo enzimático (Pectinex Ultra SP-L, Novozym 33095 e Viscozyme L), a concentração de enzima, o tempo/temperatura de incubação, pH, e a relação da atividade antimicrobiana de enzimas contra os patógenos orais relacionados à cárie dentária e doenças periodontais foram determinados pelo ensaio de difusão em disco. As condições ótimas para a extração enzimática de P. mume foram pH 6.0, 45°C, 20 h, e 5 v/v% com Pectinex Ultra SP-L. A máxima atividade antimicrobiana do extrato de P. mume obtida usando Novozym 33095 foi em pH 7.0, 45°C, 20 h, e 5 v/v%. O extrato de Viscozyme L apresentou o efeito inibitório máximo em pH 6.0, 45°C, 20 h, e 5 v/v%. O uso de combinações de enzimas não resultou em uma atividade antimicrobiana significativamente diferente (p < 0.05) em comparação com cada enzima por separada. Os valores mínimos da concentração inibitória foram de 3.125 a 12.50%. Estes resultados indicaram que a extração enzimática de alta pressão produziu o extrato de P. mume com atividade antimicrobiana,o qual tem o potencial para melhorar o ambiente bucal.
Assuntos
Bactérias , Testes de Sensibilidade Microbiana , Prunus , Compostos Fitoquímicos , Anti-InfecciososRESUMO
The late-stage doxorubicin biosynthesis pathway acting enzyme (DoxA) from Streptomyces peucetius CYP129A2 exhibited substrate promiscuity towards the stilbene group of compounds such as resveratrol. DoxA along with two accessory enzymes ferrdoxin reductase and ferredoxin from spinach hydroxylated resveratrol at the 3'-position in vitro to produce piceatannol. The product was identified by HPLC-PDA and high-resolution HR-qTOF-ESI/MS analyses in positive mode. The ESI/MS fragments resembled the hydroxylated product of resveratrol.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Estilbenos/metabolismo , Streptomyces/enzimologia , Apigenina/química , Apigenina/metabolismo , Proteínas de Bactérias/isolamento & purificação , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Doxorrubicina/biossíntese , Ferredoxinas/metabolismo , Flavanonas/química , Flavanonas/metabolismo , Flavonas/química , Flavonas/metabolismo , Hidroxilação , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Oxirredução , Conformação Proteica , Resveratrol , Estilbenos/química , Streptomyces/metabolismo , Especificidade por SubstratoRESUMO
Enzymatic alkane hydroxylation reactions are useful for producing pharmaceutical and agricultural chemical intermediates from hydrocarbons. Several cytochrome P450 enzymes catalyze the regio- and stereo-specific hydroxylation of alkanes. We evaluated the substrate binding of a putative CYP alkane hydroxylase (CYP153D17) from the bacterium Sphingomonas sp. PAMC 26605. Substrate affinities to C10-C12 n-alkanes and C10-C14 fatty acids with Kd values varied from 0.42 to 0.59 µM. A longer alkane (C12) bound more strongly than a shorter alkane (C10), while shorter fatty acids (C10, capric acid; C12, lauric acid) bound more strongly than a longer fatty acid (C14, myristic acid). These data displayed a broad substrate specificity of CYP153D17, hence it was named as a putative CYP alkane hydroxylase. Moreover, the crystal structure of CYP153D17 was determined at 3.1 Å resolution. This is the first study to provide structural information for the CYP153D family. Structural analysis showed that a co-purified alkane-like compound bound near the active-site heme group. The alkane-like substrate is in the hydrophobic pocket containing Thr74, Met90, Ala175, Ile240, Leu241, Val244, Leu292, Met295, and Phe393. Comparison with other CYP structures suggested that conformational changes in the ß1-ß2, α3-α4, and α6-α7 connecting loop are important for incorporating the long hydrophobic alkane-like substrate. These results improve the understanding of the catalytic mechanism of CYP153D17 and provide valuable information for future protein engineering studies.
Assuntos
Citocromo P-450 CYP4A/química , Citocromo P-450 CYP4A/metabolismo , Sphingomonas/enzimologia , Cristalografia por Raios X , Conformação Molecular , Ligação Proteica , Especificidade por SubstratoRESUMO
We report the complete genome sequence of Burkholderia sp. PAMC28687, which was isolated from the Antarctica lichen Useea sp., for better understanding of its catabolic traits in utilizing octopine as a source of carbon/nitrogen between Burkholderia and lichen. The genome consists of three circular chromosomes with five circular plasmids for the total 6,881,273bp sized genome with a G+C content of 58.14%.
Assuntos
Arginina/análogos & derivados , Burkholderia/genética , Burkholderia/isolamento & purificação , Genoma Bacteriano , Líquens/microbiologia , Regiões Antárticas , Arginina/metabolismo , Sequência de Bases , Análise de Sequência de DNARESUMO
Here, we report the first complete genome sequence of Frondihabitans sp. strain PAMC28766, which was found to consist of three plasmids, one chromosome (4,345,897bp), and a series of genes involved in carotenoid biosynthesis and nucleotide excision repair. An analysis of the Frondihabitans sp. PAMC28766 genome will improve our understanding of the carotenoid biosynthesis pathway. Furthermore, the sequence data will provide novel insight into UV radiation-resistance in extremely cold environments.
Assuntos
Actinomycetales/genética , Actinomycetales/isolamento & purificação , Carotenoides/biossíntese , Genoma Bacteriano , Líquens/microbiologia , Tolerância a Radiação/genética , Regiões Antárticas , Sequência de Bases , Cromossomos Bacterianos/genéticaRESUMO
In this study, we tried to characterize Streptomyces peucetius CYP157C4 with homology modeling using three cytochrome P450 (CYP) structures (CYP157C1, CYP164A2, and CYP107L1), having discovered that CYP157C4 lacks the ExxR motif that was considered invariant in all CYPs. We used Discovery Studio 3.5 to build our model after first assessing the stereochemical quality and side-chain environment, and a 7-ethoxycoumarin substrate was docked into the final model. The model-substrate complex allowed us to identify functionally important residues and validate the active-site architecture. We found a distance of 4.56 Å between the 7-ethoxycoumarin and the active site of the heme, and cloning and an in vitro assay of the CYP157C4 showed the dealkylation of the substrate. Since the details regarding this group of CYP structures are still unknown, the findings of this study may provide elucidation to assist with future efforts to find a legitimate substrate.