scholarly journals Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol

2012 ◽  
Vol 79 (5) ◽  
pp. 1619-1628 ◽  
Author(s):  
Michihiro Ito ◽  
Ikuo Sato ◽  
Masumi Ishizaka ◽  
Shin-ichiro Yoshida ◽  
Motoo Koitabashi ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Genomic Library ◽  
Enzyme System ◽  
Head Blight ◽  
Wheat Seedlings ◽  
Content Type ◽  
Cytochrome P450 System ◽  
Redox Partner ◽  
Catabolic Genes

ABSTRACTDeoxynivalenol (DON) is a natural toxin of fungi that causeFusariumhead blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON inSphingomonassp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 geneddnAwas cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent toddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstitutedin vitroin an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (kcat/Km) of 6.4 mM−1s−1. The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat.

scholarly journals Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase

2015 ◽  
Vol 81 (10) ◽  
pp. 3379-3386 ◽  
Author(s):  
Claire L. Price ◽  
Andrew G. S. Warrilow ◽  
Josie E. Parker ◽  
Jonathan G. L. Mullins ◽  
W. David Nes ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Mycosphaerella Graminicola ◽  
Temperature Sensitive ◽  
Cytochrome P450 Reductase ◽  
Zymoseptoria Tritici ◽  
Content Type ◽  
P450 Reductase ◽  
Redox Partner

ABSTRACTMycosphaerella graminicola(Zymoseptoria tritici) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14α-demethylase (CYP51), which requires cytochrome P450 reductase (CPR) as its redox partner for functional activity. TheM. graminicolaCPR (MgCPR) is able to catalyze the sterol 14α-demethylation of eburicol and lanosterol when partnered withCandida albicansCYP51 (CaCYP51) and that of eburicol only withM. graminicolaCYP51 (MgCYP51). The availability of the functionalin vivoredox partner enabled thein vitrocatalytic activity of MgCYP51 to be demonstrated for the first time. MgCYP51 50% inhibitory concentration (IC50) studies with epoxiconazole, tebuconazole, triadimenol, and prothioconazole-desthio confirmed that MgCYP51 bound these azole inhibitors tightly. The characterization of the MgCPR/MgCYP51 redox pairing has produced a functional method to evaluate the effects of agricultural azole fungicides, has demonstrated eburicol specificity in the activity observed, and supports the conclusion that prothioconazole is a profungicide.

scholarly journals Direct Electron Transfer between the frhAGB-Encoded Hydrogenase and Thioredoxin Reductase in the Nonmethanogenic Archaeon Thermococcus onnurineus NA1

2020 ◽  
Vol 86 (6) ◽  
Author(s):  
Hae-Chang Jung ◽  
Jae Kyu Lim ◽  
Tae-Jun Yang ◽  
Sung Gyun Kang ◽  
Hyun Sook Lee
Keyword(s):  
Electron Transfer ◽  
Electron Donor ◽  
Direct Electron Transfer ◽  
Thioredoxin Reductase ◽  
Thermococcus Onnurineus Na1 ◽  
Content Type ◽  
Protein Target ◽  
Redox Partner ◽  
Significant Step

ABSTRACT To date, NAD(P)H, ferredoxin, and coenzyme F420 have been identified as electron donors for thioredoxin reductase (TrxR). In this study, we present a novel electron source for TrxR. In the hyperthermophilic archaeon Thermococcus onnurineus NA1, the frhAGB-encoded hydrogenase, a homolog of the F420-reducing hydrogenase of methanogens, was demonstrated to interact with TrxR in coimmunoprecipitation experiments and in vitro pulldown assays. Electrons derived from H2 oxidation by the frhAGB-encoded hydrogenase were transferred to TrxR and reduced Pdo, a redox partner of TrxR. Interaction and electron transfer were observed between TrxR and the heterodimeric hydrogenase complex (FrhAG) as well as the heterotrimeric complex (FrhAGB). Hydrogen-dependent reduction of TrxR was 7-fold less efficient than when NADPH was the electron donor. This study not only presents a different type of electron donor for TrxR but also reveals new functionality of the frhAGB-encoded hydrogenase utilizing a protein as an electron acceptor. IMPORTANCE This study has importance in that TrxR can use H2 as an electron donor with the aid of the frhAGB-encoded hydrogenase as well as NAD(P)H in T. onnurineus NA1. Further studies are needed to explore the physiological significance of this protein. This study also has importance as a significant step toward understanding the functionality of the frhAGB-encoded hydrogenase in a nonmethanogen; the hydrogenase can transfer electrons derived from oxidation of H2 to a protein target by direct contact without the involvement of an electron carrier, which is distinct from the mechanism of its homologs, F420-reducing hydrogenases of methanogens.

scholarly journals Aphid Infestation Increases Fusarium langsethiae and T-2 and HT-2 Mycotoxins in Wheat

2016 ◽  
Vol 82 (22) ◽  
pp. 6548-6556 ◽  
Author(s):  
Jassy Drakulic ◽  
Olubukola Ajigboye ◽  
Ranjan Swarup ◽  
Toby Bruce ◽  
Rumiana V. Ray
Keyword(s):  
Aphid Transmission ◽  
Fungal Species ◽  
Head Blight ◽  
Wheat Seedlings ◽  
Aphid Infestation ◽  
Content Type ◽  
Fusarium Langsethiae ◽  
Contamination Levels ◽  
Pathogen Dna ◽  
Aphid Populations

ABSTRACTFusarium langsethiaeis a fungal pathogen of cereal crops that is an increasing problem in northern Europe, but much of its epidemiology is poorly understood. The species produces the mycotoxins T-2 and HT-2, which are highly toxic. It was hypothesized that grain aphids,Sitobion avenae, may transmitF. langsethiaeinoculum between wheat plants, and a series of transmission experiments and volatile chemical analyses was performed to test this. Manual translocation of aphids from inoculated to uninfected hosts resulted in pathogen DNA accumulation in hosts. However, the free movement of wingless aphids from infected to healthy plants did not. The addition of winged aphids reared onF. langsethiae-inoculated wheat seedlings to wheat plants also did not achieve successful pathogen transfer. While our data suggested that aphid transmission of the pathogen was not very efficient, we observed an increase in disease when aphids were present. After seedling inoculation, an increase in pathogen DNA accumulation in seedling leaves was observed upon treatment with aphids. Furthermore, the presence of aphids on wheat plants withF. langsethiae-inoculated ears not only led to a rise in the amount ofF. langsethiaeDNA in infected grain but also to an increase in the concentrations of T-2 and HT-2 toxins, with more than 3-fold higher toxin levels than diseased plants without aphids. This work highlights that aphids increase the susceptibility of wheat host plants toF. langsethiaeand that aphid infestation is a risk factor for accumulating increased levels of T-2 and HT-2 in wheat products.IMPORTANCEFusarium langsethiaeis shown here to cause increased contamination levels of grain with toxins produced by fungus when aphids share the host plant. This effect has also recently been demonstrated withFusarium graminearum, yet the two fungal species show stark differences in their effect on aphid populations. In both cases, aphids improve the ability of the pathogens to cause and initiateFusariumhead blight (FHB) disease in wheat, butF. langsethiaemay be able to act as a dispersal agent.F. langsethiaecontributes harmful toxins to wheat grain that need to be controlled, but as yet, its epidemiology is unresolved. This work reveals insights into the role aphids play in promoting the successful colonization of this species in wheat and the benefit of controlling aphid populations on crops that are at high risk of FHB.

scholarly journals An In Vitro Enzyme System for the Production of myo-Inositol from Starch

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Tomoko Fujisawa ◽  
Shohei Fujinaga ◽  
Haruyuki Atomi
Keyword(s):  
Enzyme System ◽  
Scale Production ◽  
Inositol Monophosphatase ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts ◽  
Conversion Rates ◽  
Maltodextrin Phosphorylase ◽  
Phosphate Synthase

ABSTRACT We developed an in vitro enzyme system to produce myo-inositol from starch. Four enzymes were used, maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase (MIPS), and inositol monophosphatase (IMPase). The enzymes were thermostable: MalP and PGM from the hyperthermophilic archaeon Thermococcus kodakarensis, MIPS from the hyperthermophilic archaeon Archaeoglobus fulgidus, and IMPase from the hyperthermophilic bacterium Thermotoga maritima. The enzymes were individually produced in Escherichia coli and partially purified by subjecting cell extracts to heat treatment and removing denatured proteins. The four enzyme samples were incubated at 90°C with amylose, phosphate, and NAD+, resulting in the production of myo-inositol with a yield of over 90% at 2 h. The effects of varying the concentrations of reaction components were examined. When the system volume was increased and NAD+ was added every 2 h, we observed the production of 2.9 g myo-inositol from 2.9 g amylose after 7 h, achieving gram-scale production with a molar conversion of approximately 96%. We further integrated the pullulanase from T. maritima into the system and observed myo-inositol production from soluble starch and raw potato with yields of 73% and 57 to 61%, respectively. IMPORTANCE myo-Inositol is an important nutrient for human health and provides a wide variety of benefits as a dietary supplement. This study demonstrates an alternative method to produce myo-inositol from starch with an in vitro enzyme system using thermostable maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase, and myo-inositol monophosphatase. By utilizing MalP and PGM to generate glucose 6-phosphate, we can avoid the addition of phosphate donors such as ATP, the use of which would not be practical for scaled-up production of myo-inositol. myo-Inositol was produced from amylose on the gram scale with yields exceeding 90%. Conversion rates were also high, producing over 2 g of myo-inositol within 4 h in a 200-ml reaction mixture. By adding a thermostable pullulanase, we produced myo-inositol from raw potato with yields of 57 to 61% (wt/wt). The system developed here should provide an attractive alternative to conventional methods that rely on extraction or microbial production of myo-inositol.

scholarly journals CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2

2016 ◽  
Vol 82 (22) ◽  
pp. 6507-6517 ◽  
Author(s):  
Birgit Unterweger ◽  
Dieter M. Bulach ◽  
Judith Scoble ◽  
David J. Midgley ◽  
Paul Greenfield ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Cytochrome P450 Monooxygenases ◽  
Content Type ◽  
E Coli ◽  
P450 Monooxygenases ◽  
Isolation And Characterization ◽  
Starting Point

ABSTRACTWe report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived fromSphingobium yanoikuyaeB2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6tagged) inEscherichia coliBL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation inE. colidemonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners fromE. colito yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found inNovosphingobium aromaticivoransandPseudomonas putida. Compared to P450cin(CYP176A1), a 1,8-cineole-hydroxylating P450 fromCitrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications.IMPORTANCECYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases fromS. yanoikuyaeB2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101 family. This could eventually provide enough bacterial parental enzymes with similar amino acid sequences to enablein vitroevolution via DNA shuffling.

scholarly journals Studies on the Antileishmanial Mechanism of Action of the Arylimidamide DB766: Azole Interactions and Role of CYP5122A1

2014 ◽  
Vol 58 (8) ◽  
pp. 4682-4689 ◽  
Author(s):  
Trupti Pandharkar ◽  
Xiaohua Zhu ◽  
Radhika Mathur ◽  
Jinmai Jiang ◽  
Thomas D. Schmittgen ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Mechanism Of Action ◽  
Leishmania Donovani ◽  
Synergistic Activity ◽  
Wild Type ◽  
Content Type ◽  
Axenic Amastigotes ◽  
Intracellular Parasites

ABSTRACTArylimidamides (AIAs) are inspired by diamidine antimicrobials but show superior activity against intracellular parasites. The AIA DB766 {2,5-bis[2-(2-i-propoxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride} displays outstanding potency against intracellularLeishmaniaparasites and is effective in murine and hamster models of visceral leishmaniasis when given orally, but its mechanism of action is unknown. In this study, through the use of continuous DB766 pressure, we raisedLeishmania donovaniaxenic amastigotes that displayed 12-fold resistance to this compound. These DB766-resistant (DB766R) parasites were 2-fold more sensitive to miltefosine than wild-type organisms and were hypersensitive to the sterol 14α-demethylase (CYP51) inhibitors ketoconazole and posaconazole (2,000-fold more sensitive and over 12,000-fold more sensitive than the wild type, respectively). Western blot analysis of DB766R parasites indicated that while expression of CYP51 is slightly increased in these organisms, expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism inLeishmania, is dramatically reduced in DB766R parasites.In vitrosusceptibility assays demonstrated that CYP5122A1 half-knockoutL. donovanipromastigotes were significantly less susceptible to DB766 and more susceptible to ketoconazole than their wild-type counterparts, consistent with observations in DB766R parasites. Further, DB766-posaconazole combinations displayed synergistic activity in both axenic and intracellularL. donovaniamastigotes. Taken together, these studies implicate CYP5122A1 in the antileishmanial action of the AIAs and suggest that DB766-azole combinations are potential candidates for the development of synergistic antileishmanial therapy.

scholarly journals AidC, a NovelN-Acylhomoserine Lactonase from the Potato Root-Associated Cytophaga-Flavobacteria-Bacteroides (CFB) Group Bacterium Chryseobacterium sp. Strain StRB126

2012 ◽  
Vol 78 (22) ◽  
pp. 7985-7992 ◽  
Author(s):  
Wen-Zhao Wang ◽  
Tomohiro Morohoshi ◽  
Nobutaka Someya ◽  
Tsukasa Ikeda
Keyword(s):  
High Performance ◽  
Genomic Library ◽  
Root Surface ◽  
Signal Molecules ◽  
Gram Negative Bacteria ◽  
Potato Root ◽  
Gene Encoding ◽  
Content Type ◽  
Degrading Enzymes

ABSTRACTN-Acylhomoserine lactones (AHLs) are used as quorum-sensing (QS) signal molecules by many Gram-negative bacteria. We have reported thatChryseobacteriumsp. strain StRB126, which was isolated from the root surface of potato, has AHL-degrading activity. In this study, we cloned and characterized theaidCgene from the genomic library of StRB126. AidC has AHL-degrading activity and shows homology to several metallo-β-lactamase proteins fromBacteroidetes, although not to any known AHL-degrading enzymes. Purified AidC, as a maltose-binding fusion protein, showed high degrading activity against all tested AHLs, whether short- or long-chain forms, with or without substitution at carbon 3. High-performance liquid chromatography (HPLC) analysis revealed that AidC functions as an AHL lactonase catalyzing AHL ring opening by hydrolyzing lactones. An assay to determine the effects of covalent and ionic bonding showed that Zn2+is important to AidC activity bothin vitroandin vivo. In addition, theaidCgene could also be PCR amplified from several otherChryseobacteriumstrains. In conclusion, this study indicated that theaidCgene, encoding a novel AHL lactonase, may be widespread throughout the genusChryseobacterium. Our results extend the diversity and known bacterial hosts of AHL-degrading enzymes.

scholarly journals Metabolic Drug-Drug Interaction Potential of Macrolactin A and 7-O-Succinyl Macrolactin A Assessed by Evaluating Cytochrome P450 Inhibition and Induction and UDP-Glucuronosyltransferase InhibitionIn Vitro

2014 ◽  
Vol 58 (9) ◽  
pp. 5036-5046 ◽  
Author(s):  
Soo Hyeon Bae ◽  
Min Jo Kwon ◽  
Jung Bae Park ◽  
Doyun Kim ◽  
Dong-Hee Kim ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Drug Interactions ◽  
Antitumor Agents ◽  
Content Type ◽  
Dependent Inactivation ◽  
Competitive Inhibitors ◽  
Macrolactin A ◽  
Clinically Significant

ABSTRACTMacrolactin A (MA) and 7-O-succinyl macrolactin A (SMA), polyene macrolides containing a 24-membered lactone ring, show antibiotic effects superior to those of teicoplanin against vancomycin-resistant enterococci and methicillin-resistantStaphylococcus aureus. MA and SMA are currently being evaluated as antitumor agents in preclinical studies in Korea. We evaluated the potential of MA and SMA for the inhibition or induction of human liver cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGTs)in vitroto assess their safety as new molecular entities. We demonstrated that MA and SMA are potent competitive inhibitors of CYP2C9, withKivalues of 4.06 μM and 10.6 μM, respectively. MA and SMA also weakly inhibited UGT1A1 activity, withKivalues of 40.1 μM and 65.3 μM, respectively. However, these macrolactins showed no time-dependent inactivation of the nine CYPs studied. In addition, MA and SMA did not induce CYP1A2, CYP2B6, or CYP3A4/5. On the basis of anin vitro-in vivoextrapolation, our data strongly suggested that MA and SMA are unlikely to cause clinically significant drug-drug interactions mediated via inhibition or induction of most of the CYPs involved in drug metabolismin vivo, except for the inhibition of CYP2C9 by MA. Similarly, MA and SMA are unlikely to inhibit the activity of UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 enzymesin vivo. Although further investigations will be required to clarify thein vivointeractions of MA with CYP2C9-targeted drugs, our findings offer a clearer understanding and prediction of drug-drug interactions for the safe use of MA and SMA in clinical practice.

Comparative Analysis of Three Pairs of Surrogate Redox Partners for in Vitro Activity Reconstitution of Class I Cytochrome P450 Enzymes

2021 ◽  
Author(s):  
Xiaohui Liu ◽  
Fengwei Li ◽  
Tianjian Sun ◽  
Jiawei Guo ◽  
Xingwang Zhang ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Experimental Studies ◽  
Class I ◽  
Cytochrome P450 Enzymes ◽  
Catalytic Systems ◽  
Redox Partner ◽  
Redox Partners ◽  
Lowering Drug ◽  
Electron Transfer Properties

Abstract Cytochrome P450 enzymes (P450s) are highly attractive biocatalysts due to their versatile catalytic activities. A vast majority of P450s require redox partner (RP) proteins to sequentially transfer two electrons for O2 activation and substrate oxidation. However, little information is available on cognate RPs for P450s, which greatly limits P450 function exploration and practical application. Thus, the stategy of building various hybrid P450 catalytic systems with surrogate RPs has often adopted to engineer P450 biocatalysts for different purposes. In this study, we comprehensively compare three pairs of frequently-used surrogate redox partners SelFdx1499/SelFdR0978, Adx/AdR and Pdx/PdR and in terms of their electron transfer properties. The three selected bacterial Class I P450s to accept electrons from RPs include PikC, P450sca-2 and CYP-sb21, which are responsible for production of macrolide antibiotics, the cholesterol-lowering drug pravastatin, and a hair-growth-stimulating agent. Both experimental studies and structural analysis show that SelFdx1499/SelFdR0978 is the most promising RP compared to Adx/AdR and Pdx/PdR. The results provide insights into the domination for P450-redox partner interactions in modulating the catalytic activity of P450s. This study not only produces a more active biocatalyst but also suggests a general chose for a universal reductase which would facilitate engineering of P450 catalyst.

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