Biotechnology Journal
·
2024
·Vol. 19
·期 11
·e202400518
·
IF 3.2
Fangzhi Deng,Zhenru Zhou,Zhen Du,Mohamed Mohany,Qunyue Wu,Weiyang Liang,Lei Zhang,Shan Li
Murideoxycholic acid (MDCA), as a significant secondary bile acid derived from the metabolism of α/β-muricholic acid in rodents, is an important component in maintaining the bile acid homeostasis. However, the biosynthesis of MDCA remains a challenging task. Here, we present the development of cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium , employing semi-rational protein engineering technique. Following three rounds of mutagenesis, a triple variant (T260G/G328A/L82V) has been discovered that proficiently catalyzes the 6β-hydroxylation of lithocholic acid (LCA), thereby generating MDCA with an impressive 8.5-fold increase in yield compared to the template P450 BM3 mutant. The MDCA selectivity has been also promoted from 62.0% to 96.3%. This biocatalyst introduces a novel approach for the biosynthesis of MDCA from LCA. Furthermore, molecular docking and dynamics simulations have been employed to unravel the molecular mechanisms underlying the enhanced LCA conversion and MDCA selectivity.
研究领域:
生物化学
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY
·
2022
·
IF 4.966
Ting Xiong,Si-quan Ling,Jia-li Liu,Xin-nian Zeng
The red imported fire ant ( Solenopsis invicta ), a worldwide invasive and polyphagous pest, and often nests in residential areas. Finding an alternative pesticide that is both effective on S. invicta and environmentally friendly is urgent and crucial. Fluralaner, a novel isoxazoline insecticide, has been proven to possess selective toxicity for insects versus mammals and has been safe for mammals and non-target organisms, suggesting its potential in pest management. However, little toxicity information is available for the controlment of S. invicta. In this article, we studied the toxicity of fluralaner against S. invicta systematically, and the roles of metabolism-related enzymes in the metabolism process of fluralaner. The toxicity results showed that the topical application and feeding application were all effective for S. invicta . Moreover, fluralaner can be transmitted among workers by contacting and feeding which leads to a toxic reaction among nestmates. By exploring the biochemistry change, we found cytochrome P450 monooxygenase ( P450 ) may be involved in the detoxification of fluralaner as well as carboxylesterase ( CarE ), but not glutathione S-transferase ( GST ). Synergism assays gave solid evidence in which piperonyl butoxide , an activity inhibitor of P450, increased the toxicity of fluralaner to S. invicta . Importantly, with the RNAi treatment, four of S.invicta P450 genes were significantly inhibited and showed more sensitivity to fluralaner at LC 50 concentration. Our result indicated that fluralaner could be a potential alternative pesticide in S. invicta control . And CYP9AS16 , CYP6AS161 , CYP6SQ20 , and CYP336A45 genes were closely associated with the metabolism process of fluralaner.
研究领域:
昆虫学
·
毒理学
·
生物化学
·
害虫管理
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Genes
·
2022
·Vol. 13
·期 11
·1956
·
IF 4.141
Jie Chen,Xin Hao,Ruina Tan,Yang Li,Bowen Wang,Jialiang Pan,Wei Ma,Ling Ma
Bursaphelenchus xylophilus(PWN) causes pine wilt disease (PWD), which is one of the most devastating pine diseases worldwide. Cytochrome P450 (CYP) catalyzes the biosynthetic metabolism of terpenoids and plays an important role in the modification of secondary metabolites in all living organisms. We investigated the molecular characteristics and biological functions ofBx-cyp29A3inB. xylophilus. The bioinformatics analysis results indicated thatBx-cyp29A3has a transmembrane domain and could dock with L(−)-carvone. The gene expression pattern indicated thatBx-cyp29A3was expressed in 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL L(−)-carvone solutions. TheBx-cyp29A3expression increased in a dose-dependent manner and peaked at 24 h of exposure when the L(−)-carvone solution concentration was 0.8 mg/mL. However, the gene expression peaked at 0.6 mg/mL after 36 h. Furthermore, RNA interference (RNAi) indicated thatBx-cyp29A3played an essential role in the response to L(−)-carvone. The mortality rates of theBx-cyp29A3knockdown groups were higher than those of the control groups in the 0.4, 0.6, 0.8, and 1.0 mg/mL carvone solutions after 24 h of exposure or 36 h of exposure. In summary, bioinformatics provided the structural characteristics and conserved sequence properties ofBx-cyp29A3and its encoded protein, which provided a target gene for the study of the P450 family ofB. xylophilus. Gene silencing experiments clarified the function ofBx-cyp29A3in the immune defense ofB. xylophilus.This study provides a basis for the screening of new molecular targets for the prevention and management ofB. xylophilus.Keywords:cytochrome P450;L(−)-carvone;Bursaphelenchus xylophilus;RNAi
研究领域:
分子生物学
·
植物病理学
·
线虫学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
DRUG METABOLISM AND DISPOSITION
·
2018
·Vol. 46
·期 4
·442-450
·
IF 3.64
Huimin Zhao,Siyuan Li,Zixin Yang,Ying Peng,Xiaohui Chen,Jiang Zheng
Erlotinib (ELT), a tyrosine kinase inhibitor, is widely used for the treatment of nonsmall cell lung cancer in clinic. Unfortunately, severe drug-induced liver injury and other adverse effects occurred during the treatment. Meanwhile, ELT has been reported to be a mechanism-based inactivator of cytochrome P450(CYPs) 3A4 and 3A5. The objectives of this study were to identify ketene intermediate of ELT and investigate the association of the acetylenic bioactivation with the enzyme inactivation caused by ELT. A ketene intermediate was detected in human microsomal incubations of ELT, using 4-bromobenzylamine as a trapping agent. CYPs 3A4 and 3A5 mainly contributed to the bioactivation of ELT. Microsomal incubation study showed that the ketene intermediate covalently modified the enzyme protein at lysine residues and destroyed the structure of heme. The vinyl and ethyl analogs of ELT showed minor enzyme inhibitory effect (less than 20%), whereas ELT inactivated more than 60% of the enzyme. The present study provided a novel bioactivation pathway of ELT and facilitated the understanding of the mechanisms of ELT-induced mechanism-based enzyme inactivation and liver injury.
研究领域:
药理学
·
毒理学
·
药物代谢
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Food Quality and Safety
·
2024
·
IF 3
Sun Yujing,Wang Yihua,Zhang Le,Ye Xingqian,Guo Jingtong
Furanocoumarins in citrus fruit can cause adverse drug interactions, however, there are few reports on furanocoumarins and drug interactions in common edible citrus cultivars except grapefruit. A UPLC-based method for ten furanocoumarins in citrus fruit was established and impacts on cytochrome P450 activity were investigated. Furanocoumarins were more abundant (2–43 times) in the peel than in the pulp. The pulp of Xiyou, Hongyou and Navel orange exhibited P450 inhibition with IC50 values of 0.63, 0.67 and 1.02 mg/mL, respectively. The peel of most varieties except Huyou and Satsuma exhibited P450 inhibition. Xiyou, Huangjinyou, Baiyou, Hongyou, Ponkan and Navel orange had IC50 values of 0.33, 0.38, 0.76, 0.35, 0.43 and 0.37 mg/mL, respectively. These findings indicate that the consumption of the pulp of popular Chinese varieties of citrus fruit except for Xiyou, Hongyou and Navel orange has a low risk of drug interactions, and use of citrus products including peel may carry a significant risk of adverse drug interactions.
研究领域:
药理学
·
食品科学
·
植物化学
✓ 已验证
产品关联
DOI
高影响因子
LANGMUIR
·
2014
·
IF 4.384
Dongmei Cui,Li Mi,Xuan Xu,Jusheng Lu,Jing Qian,Songqin Liu
Cytochrome P450 enzymes (cyt P450s) with an active center of iron protoheme are involved in most clinical drugs metabolism process. Herein, an electrochemical platform for the investigation of drug metabolism in vitro was constructed by immobilizing cytochrome P450 2D6 (CYP2D6) with cyt P450 reductase (CPR) on graphene modified glass carbon electrode. Direct and reversible electron transfer of the immobilized CYP2D6 with the direct electron transfer constant of 0.47 s–1 and midpoint potential of -0.483 V was obtained. In the presence of substrate tramadol, the electrochemical-driven CYP2D6 mediated catalytic behavior toward the conversion of tramadol to o-demethyl-tramadol was confirmed. The Michaelis–Menten constant (Kmapp) and heterogeneous reaction rate constant during the metabolism of tramadol were calculated to be 23.85 μM and 1.96 cm s–1, respectively. The inhibition effect of quinidine on CYP2D6 catalyze-cycle was also investigated. Furthermore, this system was applied to studying the metabolism of other drugs.
研究领域:
生物化学
·
药物代谢
·
电化学分析
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
·
2019
·
IF 2.705
Qianwen Wang,Bingbing Ma,Shinya Fushinobu,Chunfang Zhang,Lian-Hua Xu
Cytochrome P450 enzymes (P450 or CYP) are some of the most versatile biocatalysts , and offer advantages for oxidizing unreactive C–H bonds in mild conditions. In this study, we identified a novel cytochrome P450 154C2 from Streptomyces avermitilis and characterized its function in 2α-hydroxylation of testosterone with regio- and stereoselectivity . To investigate the efficiency of electron transfer , we conducted biotransformation using two different P450 redox partners—RhFRED (RhF reductase domain) from Rhodococcus sp. and Pdx (putidaredoxin)/Pdr (putidaredoxin reductase) from Pseudomonas putida and revealed that RhFRED was more effective than Pdx/Pdr, especially in vivo . The K m and k cat values for testosterone were estimated to be 0.16 ± 0.05 mM and 0.13 ± 0.02 min −1 , and k cat / K m was 0.81 min −1 mM −1 . We also determined the crystal structure of the substrate-free form of CYP154C2 at 1.5 Å resolution. The structure has a closed conformation, and the substrate binding pocket is narrow, which can explain the strict substrate specificity of the enzyme .
研究领域:
生物化学
·
酶学
·
药物代谢
✓ 已验证
DOI
PubMed
高影响因子
TOXICOLOGY LETTERS
·
2017
·
IF 3.858
Shuowen Tang,Ang Chen,Xiaojing Zhou,Li Zeng,Mingyao Liu,Xin Wang
Shikonin is a naphthoquinone pigment extracted from roots of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), and possesses various pharmaceutical activities, such as anti-inflammation and anti-cancer effects. In addition, shikonin as a natural red colorant for food garnishment and cosmetics ingredient is widely used in the world. However, the inhibition risk of shikonin on cytochrome P450 (CYP) remains unclear. The aim of this study was to investigate the potential inhibition of shikonin against CYP1A2, CYP2B1/6, CYP2C9/11, CYP2D1/6, CYP2E1 and CYP3A2/4 activities in human and rat liver microsomes through cocktail approach in vitro. The results demonstrated that shikonin exhibited no time-dependent inhibition of CYP activities. In human liver microsomes, shikonin was not only a mixed inhibitor of CYP1A2, CYP2B6, CYP2C9, CYP2D6 and CYP3A4, but also a competitive inhibitor of CYP2E1, with K i values no more than 7.72 μM. In rat liver microsomes, shikonin also exhibited the mixed inhibition on CYP1A2, CYP2B1, CYP2C11, CYP2D1, and the competitive inhibition on CYP2E1. Interestingly, shikonin presented an atypical kinetic inhibition of CYP3A2-mediated midazolam 1-hydroxylation in rats. In conclusion, the relatively low K i values of shikonin would have a high risk potential to cause the possible toxicity, especially drug-drug or food-drug interactions based on the potent inhibition of CYP enzymes.
研究领域:
药理学
·
毒理学
·
生物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FARADAY DISCUSSIONS
·
2024
·Vol. 252
·52-68
·
IF 3.3
Fengjie Jiang,Zihan Wang,Zhiqi Cong
Cytochrome P450 monooxygenases (P450s) are well recognized as versatile bio-oxidation catalysts. However, the catalytic functions of P450s are highly dependent on NAD(P)H and redox partner proteins. Our group has recently reported the use of a dual-functional small molecule (DFSM) for generating peroxygenase activity of P450BM3, a long-chain fatty acid hydroxylase from Bacillus megaterium. The DFSM-facilitated P450BM3 peroxygenase system exhibited excellent peroxygenation activity and regio-/enantioselectivity for various organic substrates, such as styrenes, thioanisole, small alkanes, and alkylbenzenes. Very recently, we demonstrated that the DFSM-facilitated P450BM3 peroxygenase could be switched to a peroxidase by engineering the redox-sensitive tyrosine residues in P450BM3. Given the great potential of P450 peroxidase for C–H oxyfunctionalization, we herein report scrutiny of the effect of mutating redox-sensitive residues on peroxidase activity by deeply screening all redox-sensitive residues of P450BM3, namely methionines, tryptophans, cysteines, and phenylalanines. As a result, six beneficial mutations at positions M212, F81, M112, F173, M177, and F77 were screened out from 78 constructed mutants, and significantly enhanced the peroxidase activity of P450BM3 in the presence of Im-C6-Phe, a typical DFSM molecule. Further combination of the beneficial mutations resulted in a more than 100-fold improvement in peroxidase activity compared with that of the combined parent enzyme and DFSM, comparable to or better than most natural peroxidases. In addition, mutations of redox-sensitive residues even dramatically increased, by more than 300-fold, the peroxidase activity of the starting F87A enzyme in the absence of the DFSM, despite the far lower apparent catalytic turnover number compared with the DFSM–P450 system. This study provides new insights and a potential strategy for regulating the catalytic promiscuity of P450 enzymes for multiple functional oxidations.
研究领域:
生物化学
·
酶工程
·
合成生物学
✓ 已验证
DOI
高影响因子
DRUG METABOLISM AND DISPOSITION
·
2020
·Vol. 48
·期 11
·1129-1136
·
IF 3.231
Jian Lu,Ang Chen,Xinrun Ma,Xuyang Shang,Yuanjin Zhang,Yuanqing Guo,Mingyao Liu,Xin Wang
Cytochrome P450 2J2 (CYP2J2) enzyme attracts more attention because it not only metabolizes clinical drugs but also mediates the biotransformation of important endogenous substances and the regulation of physiologic function. Although CYP2J2 is very important, few animal models are available to study its function in vivo. In particular, a CYP2J gene knockout (KO) rat model for drug metabolism and pharmacokinetics is not available. In this report, the CRISPR/Cas9 technology was used to delete rat CYP2J3/10, the orthologous genes of CYP2J2 in humans. The CYP2J3/10 KO rats were viable and fertile and showed no off-target effect. Compared with wild-type (WT) rats, the mRNA and protein expression of CYP2J3/10 in liver, small intestine, and heart of KO rats were completely absent. At the same time, CYP2J4 mRNA expression and protein expression were significantly decreased in these tissues. Further in vitro and in vivo metabolic studies of astemizole, a typical substrate of CYP2J, indicated that CYP2J was functionally inactive in KO rats. The heart function indexes of WT and KO rats were also measured and compared. The myocardial enzymes, including creatine kinase–muscle brain type (CK-MB), creatine kinase (CK), and CK-MB/CK ratio, of KO rats increased by nearly 140%, 80%, and 60%, respectively. In conclusion, this study successfully developed a new CYP2J3/10 KO rat model, which is a useful tool to study the function of CYP2J in drug metabolism and cardiovascular disease.
研究领域:
药理学
·
分子生物学
·
遗传学
✓ 已验证
DOI
PubMed
高影响因子
JOURNAL OF AQUATIC ANIMAL HEALTH
·
2014
·
IF 0.968
Zhi-Qiang Chang,Jian Li,Qian-Qian Zhai
Cytochrome P450s (CYPs) are the main catalytic enzymes for metabolism by a variety of endogenous and exogenous substrates in mammals, fish, insects, etc. We evaluated the application of a multidrug cocktail on changes in CYP1, CYP2, and CYP3 activity in Turbot Scophthalmus maximus. The probe drugs were a combination of caffeine (5 mg/kg body weight), dapsone (5 mg/kg), and chlorzoxazone (10 mg/kg). After a single intraperitoneal injection of the cocktail, the concentration of all three probe drugs in the plasma increased quickly to a peak and then decreased gradually over 24 h. Pharmacokinetic profiles of the three probe drugs were determined using a noncompartmental analysis, and the typical parameters were calculated. In the assay for CYP induction, pretreatment with rifampicin significantly reduced the typical pharmacokinetic metrics for caffeine and chlorzoxazone, but not dapsone, indicating that the activity of CYP1 and CYP2 in turbot were induced by rifampicin.Received November 25, 2013; accepted May 29, 2014.
研究领域:
水产养殖学
·
毒理学
·
药理学
✓ 已验证
DOI
PubMed
高影响因子
PEST MANAGEMENT SCIENCE
·
2022
·Vol. 79
·期 1
·125-133
·
IF 4.462
Bin Zeng,Ya-Ting Liu,Ze-Rui Feng,Fu-Rong Chen,Shun-Fan Wu,Chris Bass,Cong-Fen Gao
Background Buprofezin, an insect growth regulator, has been widely used to control brown planthopper (BPH), Nilaparvata lugens , one of the most destructive pests of rice crops in Asia. The intensive use of this compound has resulted in very high levels of resistance to buprofezin in the field, however, the underpinning mechanisms of resistance have not been fully resolved. Results Insecticide bioassays using the P450 inhibitor piperonyl butoxide significantly synergized the toxicity of buprofezin in two resistant strains of BPH (BPR and YC2017) compared to a susceptible strain (Sus), suggesting P450s play a role in resistance to this compound. Whole transcriptome profiling identified 1110 genes that were upregulated in the BPR strain compared to the Sus strain, including 13 cytochrome P450 genes, eight esterases and one glutathione S -transferase. Subsequently, qPCR validation revealed that four of the P450 genes, CYP6ER1vA , CYP6CW1 , CYP4C77 , and CYP439A1 were significantly overexpressed in both the BRP and YC2017 strains compared with the Sus strain. Further functional analyses showed that only suppression of CYP6ER1vA , CYP6CW1 , and CYP439A1 gene expression by RNA interference significantly increased the toxicity of buprofezin against BPH. However, only transgenic Drosophila melanogaster expressing CYP6ER1vA and CYP439A1 exhibited significant resistance to buprofezin. Finally, the BPR strain was found to exhibit modest but significant levels of resistance to acetamiprid, dinotefuran and pymetrozine. Conclusions Our findings provide strong evidence that the overexpression of CYP6ER1vA and CYP439A1 contribute to buprofezin resistance in BPH, and that resistance to this compound is associated with low-level resistance to acetamiprid, dinotefuran and pymetrozine. These results advance understanding of the molecular basis of BPH resistance to buprofezin and will inform the development of management strategies for the control of this highly damaging pest. © 2022 Society of Chemical Industry.
研究领域:
昆虫学
·
分子生物学
·
害虫管理
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）