Nature Communications
·
2023
·Vol. 14
·期 1
·1-14
·
IF 16.6
Pan Yunjun,Li Guobang,Liu Ruxin,Guo Jiawei,Liu Yunjie,Liu Mingyu,Zhang Xingwang,Chi Luping,Xu Kangwei,Wu Ruibo,Zhang Yuzhong,Li Yuezhong,Gao Xiang,Li Shengying
Cytochrome P450 enzymes play important roles in the biosynthesis of macrolide antibiotics by mediating a vast variety of regio- and stereoselective oxidative modifications, thus improving their chemical diversity, biological activities, and pharmaceutical properties. Tremendous efforts have been made on engineering the reactivity and selectivity of these useful biocatalysts. However, the 20 proteinogenic amino acids cannot always satisfy the requirement of site-directed/random mutagenesis and rational protein design of P450 enzymes. To address this issue, herein, we practice the semi-rational non-canonical amino acid mutagenesis for the pikromycin biosynthetic P450 enzyme PikC, which recognizes its native macrolide substrates with a 12- or 14-membered ring macrolactone linked to a deoxyamino sugar through a unique sugar-anchoring mechanism. Based on a semi-rationally designed substrate binding strategy, non-canonical amino acid mutagenesis at the His238 position enables the unnatural activities of several PikC mutants towards the macrolactone precursors without any sugar appendix. With the aglycone hydroxylating activities, the pikromycin biosynthetic pathway is rewired by the representative mutant PikC H238 p AcF carrying a p- acetylphenylalanine residue at the His238 position and a promiscuous glycosyltransferase. Moreover, structural analysis of substrate-free and three different enzyme-substrate complexes of PikC H238 p AcF provides significant mechanistic insights into the substrate binding and catalytic selectivity of this paradigm biosynthetic P450 enzyme.
研究领域:
生物化学
·
分子生物学
·
药学
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Yangzhen Liao,Min Wu,Jiahe Fan,Jingqiong Wan,Xin An,Xiaolan Li,Yuan Wei,Zhen Ouyang
1-Deoxynojirimycin (DNJ), a primary active component in mulberry leaves, has garnered significant attention due to its unique structure and notable pharmacological properties. Our previous investigations have elucidated the biosynthetic pathways of DNJ from lysine to 2-methylpiperidine. However, the hydroxylation process and its underlying mechanisms remain elusive. In this study, five CYP450s hydroxylase genes significantly correlated ( P < 0.05) with DNJ content in mulberry leaves at various time were screened through transcriptome profile. MaCYP71BG22 was first cloned and functionally characterized. This gene was shown to specifically catalyze the stereoselective hydroxylation of ( R )-2-methylpiperidine at the C4-position to produce (2 R , 4 R )-2-methylpiperidin-4-ol. In hairy roots of mulberry, overexpression of MaCYP71BG22 increased DNJ accumulation, while virus-induced gene silencing (VIGS) decreased its production. Furthermore, structural-function analysis pinpointed a critical residue, G460, in MaCYP71BG22, mutation of this residue to G460E enhanced the enzyme's catalytic efficiency. This study represents the first report of a CYP450 hydroxylase involved in the biosynthesis of piperidine alkaloids in mulberry leaves, and demonstrates that MaCYP71BG22 selectively catalyzes the C4-stereoselective hydroxylation of ( R )-2-methylpiperidine in DNJ biosynthesis. These findings further elucidate the DNJ biosynthetic pathway and provide new insights into the stereo- and regio-selective hydroxylation abilities of CYP450s hydroxylase in DNJ biosynthesis.
研究领域:
植物生物化学
·
分子生物学
·
药理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
NEW JOURNAL OF CHEMISTRY
·
2016
·Vol. 40
·期 10
·8928-8934
·
IF 3.277
Ren-Jie Li,Jian-He Xu,Yue-Cai Yin,Nicolas Wirth,Jiang-Meng Ren,Bu-Bing Zeng,Hui-Lei Yu
Developing a detailed understanding of the reactivity of self-sufficient Type IV P450 monooxygenases, four types of O-methylated substrates were designed as probes, including monoterpenes, cycloalkanes, aromatic compounds and steroids, and the efficiency of their oxyfunction was determined using a colorimetric assay which was based on the reaction between the enzymatic demethylation product, formaldehyde, and Purpald dye. The activity-based fingerprints of new P450RpMO, P450ArMO and P450CtMO (CYP116B members) indicated that CYP116B P450s preferentially oxidize substrates with aromatic components. Moreover, the hydroxylated products were detected based on the preference results. This rapid and efficient strategy, when coupled with GCMS, enables the exploration of the reactivity of other CYP116B members.
研究领域:
生物化学
·
酶学
·
代谢工程
✓ 已验证
产品关联
DOI
高影响因子
MOLECULES
·
2012
·Vol. 17
·期 9
·10994-11009
·
IF 2.386
Yunxia Zhang,Yunlan Li,Qingshan Li
The specific aims of this study were to evaluate the inhibition effect on CYP3A of di-n-butyl-di-(4-chlorobenzohydroxamato)tin (IV) (DBDCT), a tin-based complex with high antitumor activity, and the probable mechanism(s) of this action. Adult male SD rats were treated separately with natural saline (NS), lipopolysaccharide (LPS, 5 mg/kg), DBDCT (1.25, 2.5 and 5.0 mg/kg) intraperitoneally for 2 days after induction of CYP3A with dexamethasone (DEX, 100 mg/kg) for 4 days. Western blot analysis and fluorescent quantitation PCR (FQ-PCR) were conducted to determine the changes in expression of CYP3A, PXR, CAR and RXR. The biological accumulation of DBDCT and total Sn were determined by high-performance liquid chromatography (HPLC) and atomic fluorescence spectrometry (AFS). CYP450 content and CYP3A activities were significantly inhibited (p< 0.05) in DBDCT-treated rats compared with the control group, as was the expression of CYP3A (p< 0.05) at both protein and mRNA levels. In DBDCT-treated groups, the expression of PXR protein and mRNA increased, while the expression of CAR decreased. The biological accumulation of DBDCT and Sn in rat livers treated with DBDCT was high. The accumulation of DBDCT and Sn due to the inhibition of CYP3A may be involved in the mechanism of toxicity of DBDCT in rat liver.Keywords:cytochrome P450 3A (CYP3A);di-n-butyl-di-(4-chlorobenzohydroxamato)tin (IV) (DBDCT);inhibition;rat
研究领域:
药理学
·
毒理学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FOOD AND CHEMICAL TOXICOLOGY
·
2023
·
IF 4.3
Xin Wang,Guangyun Ran,Yufen Liao,Bowen Gong,Chutian Wu,Rong Tan,Ying Liu,Shiyu Zhang,Ying Peng,Weiwei Li,Jiang Zheng
Safrole (SFL) is an IARC class 2B carcinogen. To better understand the mechanism involved in SFL toxicity, we explored the potential interactions between SFL metabolites and RNA. Three guanosine adducts (G1-G3), two adenosine adducts (A1-A2), and two cytosine adducts (C1–C2) were detected by LC-MS/MS in mouse liver S9 incubations, cultured mouse primary hepatocytes, and liver tissues of mice after exposure to SFL. These adducts were chemically synthesized, and one of the guanosine adducts was structurally characterized by 1 H-NMR. Studies in vitro and in vivo showed that SFL was oxidized by cytochrome P450 enzymes to the corresponding 1′-hydroxyl metabolite which was further metabolized by sulfotransferases to form allylic sulfate esters. The formed reactive intermediate(s) subsequently reacted with bases of RNA, leading to RNA adduction, which could play a partial role in the toxicities of SFL through the alteration of RNA biochemical properties and interruption of RNA functions.
研究领域:
毒理学
·
药理学
·
分子生物学
·
生物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Frontiers in Plant Science
·
2017
·
IF 4.291
Su Ping,Guan Hongyu,Zhang Yifeng,Wang Xing,Gao Linhui,Zhao Yujun,Hu Tianyuan,Zhou Jiawei,Ma Baowei,Tu Lichan,Tong Yuru,Huang Luqi,Gao Wei
Tripterygium wilfordii produces not only ent-kaurene, which is an intermediate of gibberellin (GA) biosynthesis in flowering plants, but also 16α-hydroxy-ent-kaurane, whose physiological role has not been characterized. The two compounds are biosynthesized from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) by diterpene synthases, which have been discovered and functionally characterized in T. wilfordii. Here, we described the functional characterization of four cytochrome P450 reductases (TwCPR) and one ent-kaurene oxidase (TwKO). Four TwCPRs were found to have relatively ubiquitous expression in T. wilfordii root, stem, leaf, and flower tissues. Co-expression of both a TwCPR and TwKO in yeast showed that TwCPR3 has a slightly better activity for providing the electrons required for these reactions, indicating that TwCPR3 is more suitable for use in the functional analysis of other cytochrome P450 monooxygenases. TwKO catalyzed the three-step oxidation of the C4α methyl of the tetracyclic diterpene intermediate ent-kaurene to form ent-kaurenoic acid as an early step in GA biosynthesis. Notably, TwKO could also convert 16α-hydroxy-ent-kaurane to 16α-hydroxy-ent-kaurenoic acid, indicating an important function of 16α-hydroxy-ent-kaurane in the anti-HIV principle tripterifordin biosynthetic pathway in planta. Homology modeling and molecular docking were used to investigate the unknown crucial active amino acid residue involved in the catalytic reaction of TwKO, and one key residue (Leu387) contributed to the formation of 16α-hydroxy-ent-kaurenoic acid, most likely by forming hydrogen bonds with the hydroxyl group (-OH) of 16α-hydroxy-ent-kaurane, which laid the basis for further investigation of the multifunctional nature of KO catalysis. Also, our findings paved the way for the complete biosynthesis of the anti-HIV principle tripterifordin.
研究领域:
植物生物化学
·
酶学
·
分子生物学
·
生药学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Chinese Medicine
·
2020
·Vol. 15
·期 1
·1-18
·
IF 2.96
Qin Zifei,Jia Mengmeng,Yang Jing,Xing Han,Yin Zhao,Yao Zhihong,Zhang Xiaojian,Yao Xinsheng
Background Kang-Ai injection is widely used as an adjuvant therapy drug for many cancers, leukopenia, and chronic hepatitis B. Circulating alkaloids and saponins are believed to be responsible for therapeutic effects. However, their pharmacokinetics (PK) and excretion in vivo and the risk of drug–drug interactions (DDI) through inhibiting human cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes remain unclear.Methods PK and excretion of circulating compounds were investigated in rats using a validated ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC–MS) method. Further, the inhibitory effects of nine major compounds against eleven CYP and UGT isozymes were assayed using well-accepted specific substrate for each enzyme.Results After dosing, 9 alkaloids were found with Cmax and t1/2 values of 0.17–422.70 μmol/L and 1.78–4.33 h, respectively. Additionally, 28 saponins exhibited considerable systemic exposure with t1/2 values of 0.63–7.22 h, whereas other trace saponins could be negligible or undetected. Besides, over 90% of alkaloids were excreted through hepatobiliary and renal excretion. Likewise, astragalosides and protopanaxatriol (PPT) type ginsenosides also involved in hepatobiliary and/or renal excretion. Protopanaxadiol (PPD) type ginsenosides were mainly excreted to urine. Furthermore, PPD-type ginsenosides were extensively bound (fu-plasma approximately 1%), whereas astragalosides and PPT-type ginsenosides displayed fu-plasma values of 12.35% and 60.23–87.36%, respectively. Moreover, matrine, oxymatrine, astragaloside IV, ginsenoside Rg1, ginsenoside Re, ginsenoside Rd, ginsenoside Rc, and ginsenoside Rb1 exhibited no inhibition or weak inhibition against several common CYP and UGT enzymes IC50 values between 8.81 and 92.21 μM. Through kinetic modeling, their inhibition mechanisms towards those CYP and UGT isozymes were explored with obtained Ki values. In vitro-in vivo extrapolation showed the inhibition of systemic clearance for CYP or UGT substrates seemed impossible due to [I]/Ki no more than 0.1.Conclusion sWe summarized the PK behaviors, excretion characteristics and protein binding rates of circulating alkaloids, astragalosides and ginsenosides after intravenous Kang-Ai injection. Furthermore, weak inhibition or no inhibition towards these CYP and UGT activities could not trigger harmful DDI when Kang-Ai injection is co-administered with clinical drugs primarily cleared by these CYP or UGT isozymes.
研究领域:
药理学
·
药剂学
·
药物代谢
·
毒理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Molecular Catalysis
·
2023
·
IF 4.6
Xiangquan Qin,Yiping Jiang,Jie Chen,Fuquan Yao,Longyi Jin,Zhiqi Cong
Selective hydroxylation of C( sp 3 )–H bonds is a long-standing challenge in the organic synthesis field. We herein report an artificial P450 peroxygenase system facilitated by ω-pyridyl fatty acyl amino acids that can hydroxylate broad-spectrum alkanes, including linear and cyclic alkanes and benzylic and allylic alkane chains. Compared with the previously reported dual-functional small molecule (DFSM) with imidazole as a catalytic group (Im-DFSM), pyridyl DFSM (Pyd-DFSM) produced substantially higher total turnover numbers (TTNs) for the hydroxylation of medium-chain alkanes and cycloalkanes (C6–C8), e.g., 12.0-fold and 17.1-fold increased TTNs of P450 mutant F87A/T268V for heptane and cycloheptane , respectively. In addition, Pyd-DFSM altered the enantioselectivity of benzylic hydroxylation, e.g., it almost inverted the configuration observed in the benzylic hydroxylation of ethylbenzene and tetralin . More importantly, Pyd-DFSM showed excellent regioselectivity for allylic hydroxylation over epoxidation of terminal alkenes and cycloalkenes , e.g., up to 94% allylic hydroxylation of 1-hexene was observed. This work significantly enhances the catalytic toolbox of the DFSM-facilitated P450 peroxygenase system; it not only substantially expands the chemical space of P450 enzymes, but also provides an alternative solution for the selective hydroxylation of C( sp 3 )–H bonds.
研究领域:
有机化学
·
生物化学
·
催化
·
酶工程
✓ 已验证
DOI
高影响因子
CROP PROTECTION
·
2017
·
IF 1.834
Xuegui Wang,Qian Huang,Qiang Hao,Shuang Ran,Yaqiong Wu,Peng Cui,Jing Yang,Chunxian Jiang,Qunfang Yang
Spodoptera litura (Fabricius) is a widely distributed worldwide insect pest , particularly of vegetable crops . To determine the sensitivity of S. litura to insecticides , the toxicities of beta-cypermethrin, chlorpyrifos , emamectin benzoate, methoxyfenozide, and indoxacarb were tested from 2014 to 2016 using a diet incorporation assay on populations of S. litura collected from different districts in Sichuan Province and a susceptible strain (Lab-HN). Compared to the Lab-HN strain, field populations showed high levels of resistance to beta-cypermethrin (267.3- to 1789.2-fold). Field populations exhibited low or moderate resistance to chlorpyrifos in 2014 (8.9- to 24.4-fold) and high levels in 2016 (440.5- to 950.5-fold). All populations collected in 2014 and 2016 were susceptible or had a low or moderate level of resistance to indoxacarb (2.2- to 31.0-fold). No field populations exhibited resistance to emamectin benzoate or methoxyfenozide (0.7- to 1.5-fold and 0.9- to 1.4- fold) in 2014, whereas the resistance in 2016 increased to 29.8- to 55.2-fold and 38.1- to 59.0- fold, respectively. A synergist experiment indicated that the Lab-HN strain and field-collected population from Shuangliu in 2016 (SL16 population) to beta-cypermethrin may be related to cytochrome P450 monooxygenases (P450s), with the highest synergism ratios (SR) of piperonyl butoxide (PBO) of 34.4- and 73.3-fold, but not to carboxylesterase (CarE) or glutathione-S- transferase (GST). We detected the activities of detoxification enzymes and found that the ethoxycoumarin O -deethylase activities were also the strongest in the Lab-HN strain and the SL16 population with the highest SR values of 2.6- and 4.1-fold, respectively. Non-denaturing polyacrylamide gel electrophoresis (native PAGE) indicated that the inhibition of in vivo P450 activity caused by PBO was due to the binding of the synergist to the E5 or E6 isozyme , which were absent in the Lab-HN strain.
研究领域:
昆虫学
·
害虫管理
·
毒理学
·
生物化学
✓ 已验证
产品关联
DOI
高影响因子
JOURNAL OF PHARMACEUTICAL SCIENCES
·
2021
·
IF 3.534
Zifei Qin,Peile Wang,Shuyi Duan,Xiaoying Wan,Han Xing,Jing Yang,Xiaojian Zhang,Zhihong Yao,Xinsheng Yao
Isobavachalcone, a naturally occurring chalcone in Psoralea corylifolia , posses many biological properties including anticancer, antiplatelet, and antifungal. However, its glucuronidation, glucuronides excretion, and drug-drug interaction (DDI) involving in human cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT) enzymes, and efflux transporters (BCRP and MRPs) remains unclear so far. After incubation, three glucuronides were produced by HLM and HIM with total intrinsic clearance ( CL int ) of 236.71 and 323.40 μL/min/mg, respectively. Reaction phenotyping proved UGT1A1, 1A3, 1A7, 1A8, and 1A9 played important roles in glucuronidation with total CL int values of 62.69–143.00 μL/min/mg. Activity correlation analysis indicated UGT1A1 and UGT1A3 participated more in the glucuronidation. In addition, the glucuronidation showed marked species differences, and rabbits and dogs were probably appropriate model animals to investigate the in vivo glucuronidation. Furthermore, BCRP , MRP1, and MRP4 transporters were identified as the most important contributors to glucuronides excretion in HeLa1A1 cells based on gene silencing method. Moreover, isobavachalcone demonstrated broad-spectrum inhibitory effects against CYP2B6 , CYP2C9 , CYP2C19 , CYP2D6 , CYP2E1 , UGT1A1, UGT1A9, UGT2B7 with IC 50 values of 1.08–9.78 μM. Except CYP2B6 and CYP2D6, the calculated [I]/ K i values for other enzymes were all greater than 0.1, indicating the inhibition of systemic metabolism or elimination for these enzyme substrates seems likely. Taken together, we summarized metabolic fates of isobavachalcone including glucuronidation and efflux transport as well as inhibitory effects involving in human CYP and UGT enzymes.
研究领域:
药理学
·
生物化学
·
药学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Han Xie,Jinping Tang,Li Song,Guangyang Xu,Wei Li,Jinyu Zhu,Yawen Liu,Hao Ma,Linjun Cai,Xiao Xia Han
The crosstalk between endoplasmic reticulum and mitochondria is of significance in apoptosis, in which cytochrome b 5 (Cyt b 5 ) is thought to be a major target for cytochrome c (Cyt c ) upon its release from the mitochondria. In the absence of Cyt b 5 , the role of interactions of Cyt c with CYP-dependent monooxygenase system in apoptotic regulation was explored in this study. NADPH-dependent and Cyt c -induced formation of reactive oxygen species (ROS) and NADPH-independent Cyt c unfolding were revealed. With the aid of a CPR inhibitor and CYP antibodies, the interactions among Cyt c , cytochrome P450 reductase (CPR) and cytochrome P450 (CYP) are evidenced, which are found crucial for monooxygenase-derived ROS formation. The underlying structural basis of Cyt c −CYP complex was unveiled by molecular dynamics simulations. This study provides novel insights into how Cyt c regulates ROS formation through the interactions with CPR and CYP, and is implicated for a deeper understanding of the regulation mechanism in the mitochondria–endoplasmic reticulum apoptotic pathway.
研究领域:
生物化学
·
细胞生物学
·
医学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Food Bioscience
·
2026
·
IF 6.2
Mengyu Jiang,Zefeng Li,Junjie Zhang,Fei Wang,Xun Li,Yu Zhang
Dihydroquercetin (DHQ), a natural flavonoid with notable pharmacological properties, has faced microbial production challenges due to the poor functional expression of heterologous P450 enzymes in prokaryotic hosts. Here, we reported a P450-independent biosynthetic pathway for DHQ in Escherichia coli using naringenin as the precursor and the endogenous flavin monooxygenase EcHpaBC from E. coli as the core hydroxylase. Screening identified a flavonoid 3-hydroxylase (CsF3H) derived from the tea plant Camellia sinensis , which efficiently converted naringenin to dihydrokaempferol (DHK). Subsequently, EcHpaBC hydroxylated DHK at the 3′ position to yield DHQ, achieving an initial titer of 7.11 ± 0.45 mg/L. Semi-rational engineering of the rate-limiting EcHpaB subunit generated the optimal variant N460M, which increased the DHQ titer by 117.3%. Systematic optimization strategies—including plasmid copy number tuning, cofactor engineering, and fermentation condition optimization—ultimately enabled the engineered strain to achieve a final DHQ titer of 79.46 ± 1.42 mg/L, representing an 11-fold improvement over the unoptimized strain, corresponding to a molar yield of 0.261 mol mol −1 . To our knowledge, this titer represents the highest DHQ production achieved in E. coli through a non-P450 hydroxylase pathway. This pathway offered advantages in genetic simplicity, operational stability, and lower fermentation cost, thereby providing a scalable prokaryotic platform for flavonoid production that bypassed the redox partner requirement and folding limitations commonly associated with P450 enzymes.
✓ 已验证
DOI
高影响因子
危险品化学品经营许可证（带存储）