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Joonwon Kim, Pyung-Gang Lee, Eun-Ok Jung, Byung-Gee Kim
Self-sufficient CYP102As possess outstanding hydroxylating activity to fatty acids such as myristic acid. Other CYP102 subfamily members share substrate specificity of CYP102As, but, occasionally, unusual characteristics of its own subfamily have been found. In this study, only one self-sufficient cytochrome P450 from Streptomyces cattleya was renamed from CYP102A_scat to CYP102G4, purified and characterized. UV-Vis spectrometry pattern, FAD/FMN analysis, and protein sequence comparison among CYP102s have shown that CYP102 from Streptomyces cattleya belongs to CYP102G subfamily...
January 2018: Biochimica et Biophysica Acta
Natasha K Maddigan, Stephen G Bell
A self-sufficient CYP102 family encoding gene (Krac_9955) has been identified from the bacterium Ktedonobacter racemifer DSM44963 which belongs to the Chloroflexi phylum. The characterisation of the substrate range of this enzyme was hampered by low levels of production using E. coli. The yield and purity of the Krac9555 enzyme was improved using a codon optimised gene, the introduction of a tag and modification of the purification protocol. The heme domain was isolated and in vitro analysis of substrate binding and turnover was performed...
February 1, 2017: Archives of Biochemistry and Biophysics
Petr Pospíšil, Katja E Luxem, Maraia Ener, Jan Sýkora, Jana Kocábová, Harry B Gray, Antonín Vlček, Martin Hof
Fluorescence of 2-(N,N-dimethylamino)-6-propionylnaphthalene dyes Badan and Prodan is quenched by tryptophan in Brij 58 micelles as well as in two cytochrome P450 proteins (CYP102, CYP119) with Badan covalently attached to a cysteine residue. Formation of nonemissive complexes between a dye molecule and tryptophan accounts for about 76% of the fluorescence intensity quenching in micelles, the rest is due to diffusive encounters. In the absence of tryptophan, fluorescence of Badan-labeled cytochromes decays with triexponential kinetics characterized by lifetimes of about 100 ps, 700-800 ps, and 3 ns...
August 28, 2014: Journal of Physical Chemistry. B
Arthur T Kopylov, Victor G Zgoda, Andrew V Lisitsa, Alexander I Archakov
In this paper, we present a method for the determination of low- and ultralow copy-number proteins in biomaterials based on a combination of concentrating the protein from the sample onto cyanogen bromide-activated Sepharose 4B (via nonspecific binding of free amino groups) and MRM. The detection limit and the dependence of the MRM peak areas on the concentration of protein in the sample were determined using the proteins CYP102 and BSA, as a model system, both in solution and after their addition to human plasma...
March 2013: Proteomics
Bin Xiao, Xuguang Li, Jiangtao Yan, Xuefeng Yu, Guangtian Yang, Xiao Xiao, James W Voltz, Darryl C Zeldin, Dao Wen Wang
Cytochrome P450 (P450)-derived epoxyeicosatrienoic acids (EETs) exert well recognized vasodilatory, diuretic, and tubular fluid-electrolyte transport actions that are predictive of a hypotensive effect. The study sought to determine the improvement of hypertension and cardiac function by overexpressing P450 epoxygenases in vivo. Long-term expression of CYP102 F87V or CYP2J2 in spontaneously hypertensive rats (SHR) was mediated by using a type 8 recombinant adeno-associated virus (rAAV8) vector. Hemodynamics was measured by a Millar Instruments, Inc...
September 1, 2010: Journal of Pharmacology and Experimental Therapeutics
Li-ming Cheng, Jian-gang Jiang, Zi-yong Sun, Chen Chen, Ryan T Dackor, Darryl C Zeldin, Dao-wen Wang
AIM: To test the hypothesis that the epoxyeicosatrienoic acid (EET)-induced transactivation of EGF-R depends on the activation of metalloproteinases and the subsequent release of HB-EGF in cancer cells. METHODS: Exogenous 14,15-EET were added to four human-derived cancer cell lines Tca-8113, A549, HepG2, and MDA-MB-231, or these same cell lines were transfected with a mutant CYP epoxygenase (CYP102 F87V, an active 14,15-epoxygenase). The effects of elevated EET levels on the phosphorylation of tyrosine residues in the EGF receptor and on ERK1/2 activation were then assessed...
February 2010: Acta Pharmacologica Sinica
David C Lamb, Li Lei, Bin Zhao, Hang Yuan, Colin J Jackson, Andrew G S Warrilow, Tove Skaug, Paul J Dyson, Eric S Dawson, Steven L Kelly, David L Hachey, Michael R Waterman
The gene from Streptomyces coelicolor A3(2) encoding CYP102B1, a recently discovered CYP102 subfamily which exists solely as a single P450 heme domain, has been cloned, expressed in Escherichia coli, purified, characterized, and compared to its fusion protein family members. Purified reconstitution metabolism experiments with spinach ferredoxin, ferredoxin reductase, and NADPH revealed differences in the regio- and stereoselective metabolism of arachidonic acid compared to that of CYP102A1, exclusively producing 11,12-epoxyeicosa-5,8,14-trienoic acid in addition to the shared metabolites 18-hydroxy arachidonic acid and 14,15-epoxyeicosa-5,8,11-trienoic acid...
March 2010: Applied and Environmental Microbiology
Toshiki Furuya, Daisuke Shibata, Kuniki Kino
Cytochrome P450 (P450) open reading frames (ORFs) identified in genome sequences of Bacillus species are potential resources for new oxidation biocatalysts. Phylogenetic analysis of 29 Bacillus P450 ORFs revealed that the P450s consist of a limited number of P450 families, CYP102, CYP106, CYP107, CYP109, CYP134, CYP152, and CYP197. Previously, we identified the catalytic activities of three P450s of Bacillus subtilis towards steroids by rapid substrate screening using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS)...
November 2009: Steroids
Puneet K Chowdhary, Mussie Alemseghed, Donovan C Haines
CYP102s represent a family of natural self-sufficient fusions of cytochrome P450 and cytochrome P450 reductase found in some bacteria. One member of this family, named CYP102A1 or more traditionally P450BM-3, has been widely studied as a model of human P450 cytochromes. Remarkable detail of P450 structure and function has been revealed using this highly efficient enzyme. The recent rapid expansion of microbial genome sequences has revealed many relatives of CYP102A1, but to date only two from Bacillus subtilis have been characterized...
December 1, 2007: Archives of Biochemistry and Biophysics
Jian-Gang Jiang, Yao-Gui Ning, Chen Chen, Ding Ma, Zhen-Jun Liu, Shilin Yang, Jianfeng Zhou, Xiao Xiao, Xin A Zhang, Matthew L Edin, Jeffrey W Card, Jianing Wang, Darryl C Zeldin, Dao Wen Wang
Cytochrome P450 (CYP) epoxygenases convert arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EET), which exert diverse biological activities in a variety of systems. We previously reported that the CYP2J2 epoxygenase is overexpressed in human cancer tissues and cancer cell lines and that EETs enhance tumor growth, increase carcinoma cell proliferation, and prevent apoptosis of cancer cells. Herein, we report that CYP epoxygenase overexpression or EET treatment promotes tumor metastasis independent of effects on tumor growth...
July 15, 2007: Cancer Research
Marco Landwehr, Martina Carbone, Christopher R Otey, Yougen Li, Frances H Arnold
We report initial characterization of a synthetic family of more than 3000 cytochrome P450s made by SCHEMA recombination of 3 bacterial CYP102s. A total of 16 heme domains and their holoenzyme fusions with each of the 3 parental reductase domains were tested for activity on 11 different substrates. The results show that the chimeric enzymes have acquired significant functional diversity, including the ability to accept substrates not accepted by the parent enzymes. K-means clustering analysis of the activity data allowed the enzymes to be classified into five distinct groups based on substrate specificity...
March 2007: Chemistry & Biology
Jian Gang Jiang, Rui Juan Chen, Bin Xiao, Shilin Yang, Jia Ning Wang, Yong Wang, L Ashley Cowart, Xiao Xiao, Dao Wen Wang, Yong Xia
Endothelial nitric oxide synthase (eNOS) is a key enzyme in NO-mediated cardiovascular homeostasis and its activity is modulated by a variety of hormonal and mechanical stimuli via phosphorylation modification. Our previous study has demonstrated that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP)-dependent metabolites of arachidonic acid, could robustly up-regulate eNOS expression. However, the molecular mechanism underlying the effects of EETs on eNOS remains elusive. Particularly, whether and how EETs affect eNOS phosphorylation is unknown...
January 2007: Prostaglandins & Other Lipid Mediators
Karl-Heinz Storbeck, Pieter Swart, Sandra Graham, Amanda C Swart
To gain further insight into the structure/function relationship of cytochrome P450 side-chain cleavage (CYP11A1), this enzyme was investigated in the Cape baboon (Papio ursinus). Four constructs were cloned and characterised in non-steroidogenic mammalian COS-1 cells. Wild type recombinant baboon CYP11A1 cDNA yielded a K(m) value of 1.6 microM for 25-hydroxycholesterol. The single amino acid substitutions, I98Q and I98K resulted in a 1.7- and 2.8-fold increases in K(m) values, respectively. Conversely, the introduction of the mutation, K103A, resulted in a 1...
January 2007: Journal of Steroid Biochemistry and Molecular Biology
H M Girvan, T N Waltham, R Neeli, H F Collins, K J McLean, N S Scrutton, D Leys, A W Munro
Flavocytochrome P450 (cytochrome P450) BM3 is an intensively studied model system within the P450 enzyme superfamily, and is a natural fusion of a P450 to its P450 reductase redox partner. The fusion arrangement enables efficient electron transfer within the enzyme and a catalytic efficiency that cannot be matched in P450 systems from higher organisms. P450 BM3's potential for industrially relevant chemical transformations is now recognized, and variants with biotechnological applications have been constructed...
December 2006: Biochemical Society Transactions
Andrey Karyakin, Domantas Motiejunas, Rebecca C Wade, Christiane Jung
Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND)...
March 2007: Biochimica et Biophysica Acta
Karl H Clodfelter, David J Waxman, Sandor Vajda
Computational solvent mapping moves small organic molecules as probes around a protein surface, finds favorable binding positions, clusters the conformations, and ranks the clusters on the basis of their average free energy. Prior mapping studies of enzymes, crystallized in either substrate-free or substrate-bound form, have shown that the largest number of solvent probe clusters invariably overlaps in the active site. We have applied this method to five cytochromes P450. As expected, the mapping of two bacterial P450s, P450 cam (CYP101) and P450 BM-3 (CYP102), identified the substrate-binding sites in both ligand-bound and ligand-free P450 structures...
August 8, 2006: Biochemistry
Max J Cryle, Rocio D Espinoza, Sarah J Smith, Nicholas J Matovic, James J De Voss
Branched chain fatty acids are substrates for cytochrome P450(BM3) (CYP102) from Bacillus megaterium; oxidation of C15 and C17 iso and anteiso fatty acids by P450(BM3) leads to the formation of hydroxylated products that possess high levels of regiochemical and stereochemical purity.
June 14, 2006: Chemical Communications: Chem Comm
Sabine Eiben, Leonard Kaysser, Steffen Maurer, Katja Kühnel, Vlada B Urlacher, Rolf D Schmid
Isolated P450 monooxygenases have for long been neglected catalysts in enzyme technology. This is surprising as they display a remarkable substrate specificity catalyzing reactions, which represent a challenge for classic organic chemistry. On the other hand, many P450 monooxygenases are membrane bound, depend on rather complicated electron transfer systems and require expensive cofactors such as NAD(P)H. Their activities are low, and stability leaves much to be desired. The use of bacterial P450 monooxygenases from CYP102 family allows overcoming some of these handicaps...
August 5, 2006: Journal of Biotechnology
Yan Wang, Jia-ning Wang, Zhen-jun Liu, Xin Wei, Xiao Xiao, Dao-wen Wang
OBJECTIVE: To investigate the angiogenetic effects of endogenous and exogenous epoxyeicosatrienoic acids (EET) and the relevant signaling mechanisms involved. METHODS: Bovine aortic endothelial cells (BAEC) were incubated with synthetic EETs or infected with recombinant adeno-associated viruses (rAAV) containing CYP2C11-CYPOR, CYP2J2 or CYP102 F87V mutant to increase endogenous expression levels of EETs. BAEC proliferation measured by cell counting and chromatometry, migration assessed by transwell analysis, and capillary formation determined by chicken embryo chorioallantoic membrane assays (CAM) and tube formation tests on matrigel and angiogenesis were analysed in vivo...
December 2005: Zhonghua Xin Xue Guan Bing za Zhi
Roshan Perera, Masanori Sono, Gregory M Raner, John H Dawson
We describe herein for the first time the formation and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) holoenzyme and heme domain (BMP) at -55 degrees C using a 70/30 (v/v) glycerol/buffer cryosolvent. The choice of buffer is a crucial factor with Tris [tris(hydroxymethyl)aminomethane] buffer being significantly more effective than phosphate. The oxyferrous complexes have been characterized with magnetic circular dichroism spectroscopy and the resulting spectra compared to those of the more well-characterized oxyferrous cytochrome P450-CAM...
December 9, 2005: Biochemical and Biophysical Research Communications
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