RESUMO
Root exudates contain specialised metabolites that shape the plant's root microbiome. How host-specific microbes cope with these bioactive compounds, and how this ability affects root microbiomes, remains largely unknown. We investigated how maize root bacteria metabolise benzoxazinoids, the main specialised metabolites of maize. Diverse and abundant bacteria metabolised the major compound in the maize rhizosphere MBOA (6-methoxybenzoxazolin-2(3H)-one) and formed AMPO (2-amino-7-methoxy-phenoxazin-3-one). AMPO forming bacteria were enriched in the rhizosphere of benzoxazinoid-producing maize and could use MBOA as carbon source. We identified a gene cluster associated with AMPO formation in microbacteria. The first gene in this cluster, bxdA encodes a lactonase that converts MBOA to AMPO in vitro. A deletion mutant of the homologous bxdA genes in the genus Sphingobium, did not form AMPO nor was it able to use MBOA as a carbon source. BxdA was identified in different genera of maize root bacteria. Here we show that plant-specialised metabolites select for metabolisation-competent root bacteria. BxdA represents a benzoxazinoid metabolisation gene whose carriers successfully colonize the maize rhizosphere and thereby shape the plant's chemical environmental footprint.
Assuntos
Benzoxazinas , Raízes de Plantas , Rizosfera , Zea mays , Zea mays/microbiologia , Benzoxazinas/metabolismo , Raízes de Plantas/microbiologia , Raízes de Plantas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Família Multigênica , Microbiota/genética , Microbiologia do Solo , Sphingomonadaceae/genética , Sphingomonadaceae/metabolismo , Sphingomonadaceae/enzimologiaRESUMO
Aromatic compounds are an important source of commodity chemicals traditionally produced from fossil fuels. Aromatics derived from plant lignin can potentially be converted into commodity chemicals through depolymerization followed by microbial funneling of monomers and low molecular weight oligomers. This study investigates the catabolism of the ß-5 linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) by the bacterium Novosphingobium aromaticivorans. We used genome-wide screens to identify candidate genes involved in DC-A catabolism. Subsequent in vivo and in vitro analyses of these candidate genes elucidated a catabolic pathway composed of four required gene products and several partially redundant dehydrogenases that convert DC-A to aromatic monomers that can be funneled into the central aromatic metabolic pathway of N. aromaticivorans. Specifically, a newly identified γ-formaldehyde lyase, PcfL, opens the phenylcoumaran ring to form a stilbene and formaldehyde. A lignostilbene dioxygenase, LsdD, then cleaves the stilbene to generate the aromatic monomers vanillin and 5-formylferulate (5-FF). We also showed that the aldehyde dehydrogenase FerD oxidizes 5-FF before it is decarboxylated by LigW, yielding ferulic acid. We found that some enzymes involved in the ß-5 catabolism pathway can act on multiple substrates and that some steps in the pathway can be mediated by multiple enzymes, providing new insights into the robust flexibility of aromatic catabolism in N. aromaticivorans. A comparative genomic analysis predicted that the newly discovered ß-5 aromatic catabolic pathway is common within the order Sphingomonadales. IMPORTANCE: In the transition to a circular bioeconomy, the plant polymer lignin holds promise as a renewable source of industrially important aromatic chemicals. However, since lignin contains aromatic subunits joined by various chemical linkages, producing single chemical products from this polymer can be challenging. One strategy to overcome this challenge is using microbes to funnel a mixture of lignin-derived aromatics into target chemical products. This approach requires strategies to cleave the major inter-unit linkages of lignin to release monomers for funneling into valuable products. In this study, we report newly discovered aspects of a pathway by which the Novosphingobium aromaticivorans DSM12444 catabolizes aromatics joined by the second most common inter-unit linkage in lignin, the ß-5 linkage. This work advances our knowledge of aromatic catabolic pathways, laying the groundwork for future metabolic engineering of this and other microbes for optimized conversion of lignin into products.
Assuntos
Redes e Vias Metabólicas , Sphingomonadaceae , Sphingomonadaceae/metabolismo , Sphingomonadaceae/genética , Sphingomonadaceae/enzimologia , Redes e Vias Metabólicas/genética , Lignina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Hidrocarbonetos Aromáticos/metabolismoRESUMO
BACKGROUND: γ-Hexachlorocyclohexane (γ-HCH), an organochlorine insecticide of anthropogenic origin, is a persistent organic pollutant (POP) that causes environmental pollution concerns worldwide. Although many γ-HCH-degrading bacterial strains are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the low survival rate of the exogenous bacteria. Another strategy for the bioremediation of γ-HCH involves the use of transgenic plants expressing bacterial enzyme for γ-HCH degradation through phytoremediation. RESULTS: We generated transgenic Arabidopsis thaliana expressing γ-HCH dehydrochlroninase LinA from bacterium Sphingobium japonicum strain UT26. Among the transgenic Arabidopsis T2 lines, we obtained one line (A5) that expressed and accumulated LinA well. The A5-derived T3 plants showed higher tolerance to γ-HCH than the non-transformant control plants, indicating that γ-HCH is toxic for Arabidopsis thaliana and that this effect is relieved by LinA expression. The crude extract of the A5 plants showed γ-HCH degradation activity, and metabolites of γ-HCH produced by the LinA reaction were detected in the assay solution, indicating that the A5 plants accumulated the active LinA protein. In some A5 lines, the whole plant absorbed and degraded more than 99% of γ-HCH (10 ppm) in the liquid medium within 36 h. CONCLUSION: The transgenic Arabidopsis expressing active LinA absorbed and degraded γ-HCH in the liquid medium, indicating the high potential of LinA-expressing transgenic plants for the phytoremediation of environmental γ-HCH. This study marks a crucial step toward the practical use of transgenic plants for the phytoremediation of POPs.
Assuntos
Arabidopsis , Biodegradação Ambiental , Hexaclorocicloexano , Plantas Geneticamente Modificadas , Sphingomonadaceae , Arabidopsis/genética , Arabidopsis/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Hexaclorocicloexano/metabolismo , Sphingomonadaceae/genética , Sphingomonadaceae/metabolismo , Sphingomonadaceae/enzimologia , Poluentes do Solo/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Liases/genética , Liases/metabolismoRESUMO
Both polycyclic aromatic hydrocarbons (PAHs) and heavy metals persist in the environment and are toxic to organisms. Their co-occurrence makes any of them difficult to remove during bioremediation and poses challenges to environmental management and public health. Microorganisms capable of effectively degrading PAHs and detoxifying heavy metals concurrently are required to improve the bioremediation process. In this study, we isolated a new strain, Sphingobium sp. SJ10-10, from an abandoned coking plant and demonstrated its capability to simultaneously degrade 92.6 % of 75 mg/L phenanthrene and reduce 90 % of 3.5 mg/L hexavalent chromium [Cr(VI)] within 1.5 days. Strain SJ10-10 encodes Rieske non-heme iron ring-hydroxylating oxygenases (RHOs) to initiate PAH degradation. Additionally, a not-yet-reported protein referred to as Sphingobium chromate reductase (SchR), with low sequence identity to known chromate reductases, was identified to reduce Cr(VI). SchR is distributed across different genera and can be classified into two classes: one from Sphingobium members and the other from non-Sphingobium species. The widespread presence of SchR in those RHO-containing Sphingobium members suggests that they are excellent candidates for bioremediation. In summary, our study demonstrates the simultaneous removal of PAHs and Cr(VI) by strain SJ10-10 and provides valuable insights into microbial strategies for managing complex pollutant mixtures.
Assuntos
Biodegradação Ambiental , Cromatos , Dioxigenases , Oxirredutases , Hidrocarbonetos Policíclicos Aromáticos , Sphingomonadaceae , Sphingomonadaceae/enzimologia , Sphingomonadaceae/metabolismo , Dioxigenases/metabolismo , Dioxigenases/genética , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/química , Cromatos/metabolismo , Oxirredutases/metabolismo , Cromo/metabolismo , Fenantrenos/metabolismoRESUMO
The formation of amide bonds via aminolysis of esters by lipases generates a diverse range of amide frameworks in biosynthetic chemistry. Few lipases have satisfactory activity towards bulky aromatic amines despite numerous attempts to improve the efficiency of this transformation. Here, we report the discovery of a new intracellular lipase (Ndbn) with a broad substrate scope. Ndbn turns over a range of esters and aromatic amines in the presence of water (2 %; v/v), producing a high yield of multiple valuable amides. Remarkably, a higher conversion rate was observed for the synthesis of amides from substrates with aromatic amine rather than aliphatic amines. Molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) studies showcase the mechanism for the preference for aromatic amines, including a more suitable orientation, shorter catalytic distances in the active site pocket and a lower reaction barrier for aromatic than for aliphatic amines. This unique lipase is thus a promising biocatalyst for the efficient synthesis of aromatic amides.
Assuntos
Aminas , Ésteres , Lipase , Lipase/metabolismo , Lipase/química , Aminas/química , Ésteres/química , Simulação de Dinâmica Molecular , Especificidade por Substrato , Amidas/química , Domínio Catalítico , Biocatálise , Sphingomonadaceae/enzimologiaRESUMO
BACKGROUND: Icaritin is an aglycone of flavonoid glycosides from Herba Epimedii. It has good performance in the treatment of hepatocellular carcinoma in clinical trials. However, the natural icaritin content of Herba Epimedii is very low. At present, the icaritin is mainly prepared from flavonoid glycosides by α-L-rhamnosidases and ß-glucosidases in two-step catalysis process. However, one-pot icaritin production required reported enzymes to be immobilized or bifunctional enzymes to hydrolyze substrate with long reaction time, which caused complicated operations and high costs. To improve the production efficiency and reduce costs, we explored α-L-rhamnosidase SPRHA2 and ß-glucosidase PBGL to directly hydrolyze icariin to icaritin in one-pot, and developed the whole-cell catalytic method for efficient icaritin production. RESULTS: The SPRHA2 and PBGL were expressed in Escherichia coli, respectively. One-pot production of icaritin was achieved by co-catalysis of SPRHA2 and PBGL. Moreover, whole-cell catalysis was developed for icariin hydrolysis. The mixture of SPRHA2 cells and PBGL cells transformed 200 g/L icariin into 103.69 g/L icaritin (yield 95.23%) in 4 h in whole-cell catalysis under the optimized reaction conditions. In order to further increase the production efficiency and simplify operations, we also constructed recombinant E. coli strains that co-expressed SPRHA2 and PBGL. Crude icariin extracts were also efficiently hydrolyzed by the whole-cell catalytic system. CONCLUSIONS: Compared to previous reports on icaritin production, in this study, whole-cell catalysis showed higher production efficiency of icaritin. This study provides promising approach for industrial production of icaritin in the future.
Assuntos
Indústria Farmacêutica , Medicamentos de Ervas Chinesas , Flavonoides , Microbiologia Industrial , Catálise , Medicamentos de Ervas Chinesas/síntese química , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/metabolismo , Escherichia coli/genética , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genética , Paenibacillus/enzimologia , Paenibacillus/genética , Microbiologia Industrial/métodos , Indústria Farmacêutica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Flavonoides/biossíntese , HidróliseRESUMO
Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.
Assuntos
Hidrolases/química , Mycobacterium bovis/enzimologia , Sphingomonadaceae/enzimologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X/métodos , Evolução Molecular , Hidrolases/metabolismo , Hidrólise , Modelos Moleculares , Engenharia de Proteínas/métodos , Análise de Sequência de Proteína/métodosRESUMO
Lignin is a potential source of valuable chemicals, but its chemical depolymerization results in a heterogeneous mixture of aromatics and other products. Microbes could valorize depolymerized lignin by converting multiple substrates into one or a small number of products. In this study, we describe the ability of Novosphingobium aromaticivorans to metabolize 1-(4-hydroxy-3-methoxyphenyl)propane-1,2-dione (G-diketone), an aromatic Hibbert diketone that is produced during formic acid-catalyzed lignin depolymerization. By assaying genome-wide transcript levels from N. aromaticivorans during growth on G-diketone and other chemically-related aromatics, we hypothesized that the Lig dehydrogenases, previously characterized as oxidizing ß-O-4 linkages in aromatic dimers, were involved in G-diketone metabolism by N. aromaticivorans. Using purified N. aromaticivorans Lig dehydrogenases, we found that LigL, LigN, and LigD each reduced the Cα ketone of G-diketone in vitro but with different substrate specificities and rates. Furthermore, LigL, but not LigN or LigD, also reduced the Cα ketone of 2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one (GP-1) in vitro, a derivative of G-diketone with the Cß ketone reduced, when GP-1 was provided as a substrate. The newly identified activity of these Lig dehydrogenases expands the potential range of substrates utilized by N. aromaticivorans beyond what has been previously recognized. This is beneficial both for metabolizing a wide range of natural and non-native depolymerized lignin substrates and for engineering microbes and enzymes that are active with a broader range of aromatic compounds. IMPORTANCE Lignin is a major plant polymer composed of aromatic units that have value as chemicals. However, the structure and composition of lignin have made it difficult to use this polymer as a renewable source of industrial chemicals. Bacteria like Novosphingobium aromaticivorans have the potential to make chemicals from lignin not only because of their natural ability to metabolize a variety of aromatics but also because there are established protocols to engineer N. aromaticivorans strains to funnel lignin-derived aromatics into valuable products. In this work, we report a newly discovered activity of previously characterized dehydrogenase enzymes with a chemically modified by-product of lignin depolymerization. We propose that the activity of N. aromaticivorans enzymes with both native lignin aromatics and those produced by chemical depolymerization will expand opportunities for producing industrial chemicals from the heterogenous components of this abundant plant polymer.
Assuntos
Cetonas , Lignina , Oxirredutases/metabolismo , Sphingomonadaceae/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Microbiologia Industrial , Cetonas/metabolismo , Lignina/metabolismo , Oxirredutases/genéticaRESUMO
The reaction sequence for aerobic degradation of bile salts by environmental bacteria resembles degradation of other steroid compounds. Recent findings show that bacteria belonging to the Sphingomonadaceae use a pathway variant for bile-salt degradation. This study addresses this so-called Δ4,6-variant by comparative analysis of unknown degradation steps in Sphingobium sp. strain Chol11 with known reactions found in Pseudomonas stutzeri Chol1. Investigations of strain Chol11 revealed an essential function of the acyl-CoA dehydrogenase (ACAD) Scd4AB for growth with bile salts. Growth of the scd4AB deletion mutant was restored with a metabolite containing a double bond within the side chain which was produced by the Δ22-ACAD Scd1AB from P. stutzeri Chol1. Expression of scd1AB in the scd4AB deletion mutant fully restored growth with bile salts, while expression of scd4AB only enabled constricted growth in P. stutzeri Chol1 scd1A or scd1B deletion mutants. Strain Chol11 Δscd4A accumulated hydroxylated steroid metabolites which were degraded and activated with coenzyme A by the wild type. Activities of five Rieske type monooxygenases of strain Chol11 were screened by heterologous expression and compared to the B-ring cleaving KshABChol1 from P. stutzeri Chol1. Three of the Chol11 enzymes catalyzed B-ring cleavage of only Δ4,6-steroids, while KshABChol1 was more versatile. Expression of a fourth KshA homolog, Nov2c228, led to production of metabolites with hydroxylations at an unknown position. These results indicate functional diversity of proteobacterial enzymes for bile-salt degradation and suggest a novel side chain degradation pathway involving an essential ACAD reaction and a steroid hydroxylation step. IMPORTANCE This study highlights the biochemical diversity of bacterial degradation of steroid compounds in different aspects. First, it further elucidates an unexplored variant in the degradation of bile-salt side chains by sphingomonads, a group of environmental bacteria that is well-known for their broad metabolic capabilities. Moreover, it adds a so far unknown hydroxylation of steroids to the reactions Rieske monooxygenases can catalyze with steroids. Additionally, it analyzes a proteobacterial ketosteroid-9α-hydroxylase and shows that this enzyme is able to catalyze side reactions with nonnative substrates.
Assuntos
Acil-CoA Desidrogenase/metabolismo , Ácidos e Sais Biliares/metabolismo , Oxigenases de Função Mista/metabolismo , Pseudomonas stutzeri , Sphingomonadaceae , Esteroides/metabolismo , Proteínas de Bactérias/metabolismo , Pseudomonas stutzeri/enzimologia , Pseudomonas stutzeri/genética , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genéticaRESUMO
Nitroreductases are emerging as attractive bioremediation enzymes, with substrate promiscuity toward both natural and synthetic compounds. Recently, the nitroreductase NfnB from Sphingopyxis sp. strain HMH exhibited metabolic activity for dinitroaniline herbicides including butralin and pendimethalin, triggering the initial steps of their degradation and detoxification. However, the determinants of the specificity of NfnB for these herbicides are unknown. In this study, we performed structural and biochemical analyses of NfnB to decipher its substrate specificity. The homodimer NfnB is a member of the PnbA subgroup of the nitroreductase family. Each monomer displays a central α + ß fold for the core domain, with a protruding middle region and an extended C-terminal region. The protruding middle region of Val75-Tyr129 represents a structural extension that is a common feature to members of the PnbA subgroup and functions as an opening wall connecting the coenzyme FMN-binding site to the surface, therefore serving as a substrate binding site. We performed mutational, kinetic, and structural analyses of mutant enzymes and found that Tyr88 in the middle region plays a pivotal role in substrate specificity by determining the dimensions of the wall opening. The mutation of Tyr88 to phenylalanine or alanine caused significant changes in substrate selectivity toward bulkier dinitroaniline herbicides such as oryzalin and isopropalin without compromising its activity. These results provide a framework to modify the substrate specificity of nitroreductase in the PnbA subgroup, which has been a challenging issue for its biotechnological and bioremediation applications.
Assuntos
Compostos de Anilina/química , Dinitrobenzenos/química , Herbicidas/química , Nitrorredutases/química , Sphingomonadaceae/enzimologia , Sulfanilamidas/química , Sítios de Ligação , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Sphingorhabdus sp. YGSMI21, a novel microbial strain with an enantioselective epoxide hydrolase activity, was isolated from tidal samples contaminated by accidental oil spills subjected to enriched culture with polycyclic aromatic hydrocarbon. This strain was able to optically decompose (R)-styrene oxide (SO) and showed 100% optical purity. In addition, it showed a good enantioselectivity for the derivatives of (S)-SO, (S)-2-chlorostyrene oxide (CSO), (S)-3-CSO and (S)-4-CSO. For (S)-2-CSO, (S)-3-CSO and (S)-4-CSO, 99.9%ee was obtained with the yield of 26.2%, 24.8%, and 11.0%, respectively, when using 10 mg cells of Sphingorhabdus sp. YGSMI21 at pH 8.0 with 4 mM racemic substrates at pH 8.0 and 25°C. The values obtained in this study for (S)-2-CSO, particularly the yield of 26.2%, is noteworthy, considering that obtaining an enantiomerically pure form is difficult. Taken together, Sphingorhabdus sp. YGSMI21 can be regarded as a whole-cell biocatalyst in the production of various (S)-CSO with the chlorine group at a different position.
Assuntos
Epóxido Hidrolases/metabolismo , Compostos de Epóxi/metabolismo , Sedimentos Geológicos/microbiologia , Sphingomonadaceae/isolamento & purificação , Hidrólise , Sphingomonadaceae/classificação , Sphingomonadaceae/enzimologia , EstereoisomerismoRESUMO
1-Naphthol, a widely used raw material for organic synthesis, is also a well-known organic pollutant. Due to its high toxicity, 1-naphthol is rarely used by microorganisms as the sole carbon source for growth. In this study, catabolism of 1-naphthol by Sphingobium sp. strain B2 was found to be greatly enhanced by additional supplementation with primary carbon sources (e.g., glucose, maltose, and sucrose), and 1-naphthol was even used as the carbon source for growth when strain B2 cells had been preinduced by both 1-naphthol and glucose. A distinct two-component flavin-dependent monooxygenase, NdcA1A2, was found to be responsible for the initial hydroxylation of 1-naphthol to 1,2-dihydroxynaphthalene, a more toxic compound. Transcriptional levels of ndcA1A2 genes were significantly upregulated when strain B2 cells were cultured with both 1-naphthol and glucose compared to cells cultured with only 1-naphthol or glucose. Two transcriptional regulators, the activator NdcS and the inhibitor NdcR, were found to play key roles in the synergistic regulation of the transcription of the 1-naphthol initial catabolism genes ndcA1A2IMPORTANCE Cometabolism is a widely observed phenomenon, especially in the field of microbial catabolism of highly toxic xenobiotics. However, the mechanisms of cometabolism are ambiguous, and the roles of the obligately coexisting growth substrates remain largely unknown. In this study, we revealed that the roles of the coexisting primary carbon sources (e.g., glucose) in the enhanced catabolism of the toxic compound 1-naphthol in Sphingobium sp. strain B2 were not solely because they were used as growth substrates to support cell growth but, more importantly, because they acted as coinducers to interact with two transcriptional regulators, the activator NdcS and the inhibitor NdcR, to synergistically regulate the transcription of the 1-naphthol initial catabolism genes ndcA1A2 Our findings provide new insights into the cometabolic mechanism of highly toxic compounds in microorganisms.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Oxigenases de Função Mista/genética , Naftóis/metabolismo , Sphingomonadaceae/genética , Proteínas de Bactérias/metabolismo , Oxigenases de Função Mista/metabolismo , Sphingomonadaceae/enzimologiaRESUMO
We identified three novel microbial esterase (Est1, Est2, and Est3) from Sphingobium chungbukense DJ77. Multiple sequence alignment showed the Est1 and Est3 have distinct motifs, such as tetrapeptide motif HGGG, a pentapeptide sequence motif GXSXG, and catalytic triad residues Ser-Asp-His, indicating that the identified enzymes belong to family IV esterases. Interestingly, Est1 exhibited strong activity toward classical esterase substrates, p-nitrophenyl ester of short-chain fatty acids and long-chain. However, Est3 did not exhibit any activity despite having high sequence similarity and sharing the identical catalytic active residues with Est1. Est3 only showed hydrolytic degradation activity to polycaprolactone (PCL). MOE-docking prediction also provided the parameters consisting of binding energy, molecular docking score, and molecular distance between substrate and catalytic nucleophilic residue, serine. The engineered mutEst3 has hydrolytic activity for a variety of esters ranging from p-nitrophenyl esters to PCL. In the present study, we demonstrated that MOE-docking simulation provides a valuable insight for facilitating biocatalytic performance.
Assuntos
Clonagem Molecular/métodos , Esterases/química , Esterases/metabolismo , Poliésteres/química , Sphingomonadaceae/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Esterases/genética , Concentração de Íons de Hidrogênio , Hidrólise , Simulação de Acoplamento Molecular , Alinhamento de Sequência , Sphingomonadaceae/química , Sphingomonadaceae/genética , Especificidade por SubstratoRESUMO
Fumonisins have posed hazardous threat to human and animal health worldwide. Enzymatic degradation is a desirable detoxification approach but is severely hindered by serious shortage of detoxification enzymes. After mining enzymes by bioinformatics analysis, a novel carboxylesterase FumDSB from Sphingomonadales bacterium was expressed in Escherichia coli, and confirmed to catalyze fumonisin B1 to produce hydrolyzed fumonisin B1 by liquid chromatography mass spectrometry for the first time. FumDSB showed high sequence novelty, sharing only ~34% sequence identity with three reported fumonisin detoxification carboxylesterases. Besides, FumDSB displayed its high degrading activity at 30-40 °C within a broad pH range from 6.0 to 9.0, which is perfectly suitable to be used in animal physiological condition. It also exhibited excellent pH stability and moderate thermostability. This study provides a FB1 detoxification carboxylesterase which could be further used as a potential food and feed additive.
Assuntos
Carboxilesterase/química , Fumonisinas/química , Alphaproteobacteria/metabolismo , Animais , Carboxilesterase/isolamento & purificação , Carboxilesterase/metabolismo , Hidrolases de Éster Carboxílico/química , Cromatografia Líquida , Fumonisinas/análise , Fumonisinas/metabolismo , Humanos , Espectrometria de Massas , Sphingomonadaceae/enzimologiaRESUMO
Short-chain chlorinated paraffins (SCCPs) are listed as persistent organic pollutants (POPs) under the Stockholm Convention. Such substances are toxic, bioaccumulating, transported over long distances and degrade slowly in the environment. Certain bacterial strains of the Sphingomonadacea family are able to degrade POPs, such as hexachlorocyclohexanes (HCHs) and hexabromocyclododecanes (HBCDs). The haloalkane dehalogenase LinB, expressed in certain Sphingomonadacea, is able to catalyze the transformation of haloalkanes to hydroxylated compounds. Therefore, LinB is a promising candidate for conversion of SCCPs. Hence, a mixture of chlorinated tridecanes was exposed in vitro to LinB, which was obtained through heterologous expression in Escherichia coli. Liquid chromatography mass spectrometry (LC-MS) was used to analyze chlorinated tridecanes and their transformation products. A chloride-enhanced soft ionization method, which favors the formation of chloride adducts [M+Cl]- without fragmentation, was applied. Mathematical deconvolution was used to distinguish interfering mass spectra of paraffinic, mono-olefinic and di-olefinic compounds. Several mono- and di-hydroxylated products including paraffinic, mono-olefinic and di-olefinic compounds were found after LinB exposure. Mono- (rt = 5.9-6.9 min) and di-hydroxylated (rt = 3.2-4.5 min) compounds were separated from starting material (rt = 7.7-8.5 min) by reversed phase LC. Chlorination degrees of chlorinated tridecanes increased during LinB-exposure from nCl = 8.80 to 9.07, indicating a preferential transformation of lower chlorinated (Cl<9) tridecanes. Thus, LinB indeed catalyzed a dehalohydroxylation of chlorinated tridecanes, tridecenes and tridecadienes. The observed hydroxylated compounds are relevant CP transformation products whose environmental and toxicological effects should be further investigated.
Assuntos
Poluentes Ambientais/análise , Hidrocarbonetos Clorados/análise , Hidrolases/química , Parafina/análise , Biocatálise , Monitoramento Ambiental/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Halogenação , Hexaclorocicloexano/análise , Hidrocarbonetos Bromados/análise , Hidrolases/isolamento & purificação , Hidroxilação , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genéticaRESUMO
Four phenylacetaldehyde dehydrogenases (designated as FeaB or StyD) originating from styrene-degrading soil bacteria were biochemically investigated. In this study, we focused on the Michaelis-Menten kinetics towards the presumed native substrate phenylacetaldehyde and the obviously preferred co-substrate NAD+. Furthermore, the substrate specificity on four substituted phenylacetaldehydes and the co-substrate preference were studied. Moreover, these enzymes were characterized with respect to their temperature as well as long-term stability. Since aldehyde dehydrogenases are known to show often dehydrogenase as well as esterase activity, we tested this capacity, too. Almost all results showed clearly different characteristics between the FeaB and StyD enzymes. Furthermore, FeaB from Sphingopyxis fribergensis Kp5.2 turned out to be the most active enzyme with an apparent specific activity of 17.8 ± 2.1 U mg-1. Compared with that, both StyDs showed only activities less than 0.2 U mg-1 except the overwhelming esterase activity of StyD-CWB2 (1.4 ± 0.1 U mg-1). The clustering of both FeaB and StyD enzymes with respect to their characteristics could also be mirrored in the phylogenetic analysis of twelve dehydrogenases originating from different soil bacteria.
Assuntos
Aldeído Oxirredutases/química , Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Microbiologia do Solo , Sphingomonadaceae/enzimologia , Estireno/metabolismoRESUMO
The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is notable for its ability to hydrolyze a broad spectrum of organophosphate triesters, including organophosphorus flame retardants and plasticizers such as triphenyl phosphate and tris(2-chloroethyl) phosphate that are not substrates for other enzymes. This enzyme is also capable of hydrolyzing any one of the three ester groups attached to the central phosphorus core. The enantiomeric isomers of 1,1'-bi-2-naphthol (BINOL) have become among the most widely used chiral auxiliaries for the chemical synthesis of chiral carbon centers. PTE was tested for its ability to hydrolyze a series of biaryl phosphate esters, including mono- and bis-phosphorylated BINOL derivatives and cyclic phosphate triesters. Sb-PTE was shown to be able to catalyze the hydrolysis of the chiral phosphate triesters with significant stereoselectivity. The catalytic efficiency, kcat/Km, of Sb-PTE toward the test phosphate triesters ranged from â¼10 to 105 M-1 s-1. The product ratios and stereoselectivities were determined for four pairs of phosphorylated BINOL derivatives.
Assuntos
Naftóis/química , Hidrolases de Triester Fosfórico/metabolismo , Sphingomonadaceae/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catálise , Hidrólise , Cinética , Naftóis/metabolismo , Fosfatos/química , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/genética , Estereoisomerismo , Especificidade por SubstratoRESUMO
4-Hydroxyphenylpyruvate dioxygenase (HPPD) has attracted extensive interest as a promising target for the genetic engineering of herbicide-resistant crops. However, naturally occurring HPPDs are generally very sensitive to HPPD inhibitors. In this study, random mutagenesis was performed to increase the HPPD inhibitors' resistance of Sphingobium sp. HPPD (SpHPPD). Two mutants, Q258M and Y333F, with improved resistance were obtained. Subsequently, a double-mutant (Q258M/Y333F) was generated through combined mutation. Q258M/Y333F exhibited the highest resistance to four HPPD inhibitors [topramezone, mesotrione, tembotrione, and diketonitrile (DKN)]. The enzyme fitness of Q258M/Y333F to topramezone, mesotrione, tembotrione, and DKN was increased by 4.0-, 4.1-, 4.2-, and 3.2-folds, respectively, in comparison with that of the wild-type. Molecular modeling and docking revealed that Q258M mutation leads to the decrease of enzyme-inhibitor-binding strength by breaking the hydrogen bond between the enzyme and the inhibitor, and Y333F mutation changes the conformational balance of the C-terminal helix H11, which hinders the binding of the inhibitor to the enzyme and thus would contribute to improved herbicide resistance. This study helps to further elucidate the structural basis for herbicide resistance and provides better genetic resources for the genetic engineering of herbicide-resistant crops.
Assuntos
4-Hidroxifenilpiruvato Dioxigenase/química , 4-Hidroxifenilpiruvato Dioxigenase/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Herbicidas/química , Sphingomonadaceae/enzimologia , 4-Hidroxifenilpiruvato Dioxigenase/antagonistas & inibidores , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Evolução Molecular Direcionada , Inibidores Enzimáticos/química , Resistência a Herbicidas , Simulação de Acoplamento Molecular , Sphingomonadaceae/química , Sphingomonadaceae/genéticaRESUMO
The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via ß-(2â6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans, and Paraburkolderia graminis. The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree.
Assuntos
Frutanos/química , Frutose/química , Gluconobacter oxydans/enzimologia , Hexosiltransferases/química , Oligossacarídeos/química , Sphingomonadaceae/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Burkholderiaceae/química , Burkholderiaceae/enzimologia , Frutanos/biossíntese , Frutose/metabolismo , Expressão Gênica , Gluconobacter oxydans/química , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Oligossacarídeos/biossíntese , Prebióticos/análise , Ligação Proteica , Conformação Proteica , Rafinose/química , Rafinose/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Sphingomonadaceae/química , Homologia Estrutural de Proteína , Especificidade por Substrato , Sacarose/química , Sacarose/metabolismo , Vibrio/química , Vibrio/enzimologiaRESUMO
The amination of racemic alcohols to produce enantiopure amines is an important green chemistry reaction for pharmaceutical manufacturing, requiring simple and efficient solutions. Herein, we report the development of a cascade biotransformation to aminate racemic alcohols. This cascade utilizes an ambidextrous alcohol dehydrogenase (ADH) to oxidize a racemic alcohol, an enantioselective transaminase (TA) to convert the ketone intermediate to chiral amine, and isopropylamine to recycle PMP and NAD+ cofactors via the reversed cascade reactions. The concept was proven by using an ambidextrous CpSADH-W286A engineered from (S)-enantioselective CpSADH as the first example of evolving ambidextrous ADHs, an enantioselective BmTA, and isopropylamine. A biosystem containing isopropylamine and E.â coli (CpSADH-W286A/BmTA) expressing the two enzymes was developed for the amination of racemic alcohols to produce eight useful and high-value (S)-amines in 72-99 % yield and 98-99 % ee, providing with a simple and practical solution to this type of reaction.