IMMUNOCHEMICAL IDENTIFICATION AND LOCALIZATION OF CYTOCHROME P-450HSjISOZYME, AN ENZYME RELATED TO NITROSAMINE METABOLISM, IN HUMAN GASTRIC MUCOSA AND GASTRIC CARCINOMA

Monoclonal antibody (MAb) to rat liver cyto-chrome P-450j isozyme, an activating enzyme specific to nitrosamine metabolism, was used coupled with immunoblotting, densitometer scanning of SDS-PAGE gels and immunohistochemical technique. The trace P-450HSj isozyme (Mr. 51.5 Kd) was found in human gastric mucosa. It was similar to P-450j in molecular weight, catalytic and immunochemical properties. The concentrations of P-450HSj in mucosa of lesser curvature were higher than those in greater curvature. This might be one of the important reasons that lesser curvature is the commonest area for gastric carcinoma. But there was possibly less P-450HSj in gastric mucosa with cancer. Im-munohistochemically, P-450HSj was discovered in the cytoplasm of some glandular epithelial cells, especially in the glands with hyperplastic and intestinal metaplastic changes adjacent to carcinoma. It was also found in some normal glands and in tumor cells of high-differentiated adenocarcinoma, but not in those of low-differentiated ones. Following subjects are discussed: (1) the method of detecting trace P-450HSj, (2) the rule of distribution of P-450HSj, and (3) the relationship between the isozyme and the occurrence of gastric cancer caused by nitrosa-mines.

In vitro and in vivo cytochrome P450 3A enzyme inhibition by Aframomum melengueta and Dennettia tripetala extracts

Objective: To evaluate the in vitro and in vivo inhibitory effects of two commonly used herbs, Aframomum melengueta(A. melengueta) and Dennettia tripetala(D. tripetala) on CYP 3A enzymes. Methods: In vitro inhibition of the enzymes were assessed with microsomes extracted from female albino rats using erythromycin-N-demethylation assay(EMND) method while their in vivo effects were measured by estimating simvastatin plasma concentrations in rats. Pharmacokinetic parameters were determined using non-compartmental anaysis as implemented in Win Nonlin pharmacokinetic program. Results: EMND assay with intestinal microsomes indicated that aqueous extracts of D. tripetala and A. melengueta significantly(P < 0.05) inhibited intestinal CYP 3A activity at both 50 μg and 100 μg concentrations. Petroleum ether extract of D. tripetala and ethanol extracts of A. melengueta inhibited intestinal CYP3 A activity at 100 μg but not at 50 μg concentrations. All the extracts showed an in vitrodose dependent CYP 3A inhibition with liver microsomes. In vivo analysis showed that pretreatment with the extracts enhanced systemic absorption of simvastatin with reductions in metabolizing enzymes activity as indicated in significant increases in maximal concentration, area under curve, area under moment curve and mean resident time of simvastatin(P < 0.05). Conclusions: Herbal preparations containing these plants' extracts should be used with caution especially in patients on CYP450 3A substrate medications.

Application of Precision-Cut Rat Liver Slice to Study the Influence of Monocrotaline, Tussilago farfara Alkaloids on the Expression of Cytochrome P450 Enzymes

Precision-cut liver slice has been successfully used to study the mechanism of drug-induced hepatotoxicity, the prediction of liver toxicity, the discovery of early hepatic toxicity biomarker and the metabolism of drug in liver. We detected the expression of CYP3A4, CYP2B1 + CYP2B2 and CYP2E1 in precision-cut liver slice after co-cultured with monocrotaline or Tussilago farfara alkaloids to investigate the hepatotoxicity mechanism of those drugs. After co-culturing with monocrotaline or Tussilago farfara alkaloids for 6 hours, the expression of CYP3A4 in the microsome of precision-cut liver slices was detected by Western blot, and the expressions of CYP2B1 + CYP2B2 and CYP2E1 were detected by immunofluorescence. The results showed that monocrotaline induced the expression of CYP3A4 and CYP2B1 + CYP2B2, and Tussilago farfara alkaloids obviously up-regulated the expression of CYP2E1 and CYP3A4. Thus, we conclude that the up-regulation of CYP3A4, CYP2B1 + CYP2B2 and CYP2E1 may be one of the toxic mechanisms of liver injury of those drugs.

Cytochrome P450 Directed Prodrug Activation Therapy in Research of Cancer Enzymology

Cancer enzymology is a promising filiation of bio-medical sciences. In thepast decades, enzymes, such as GST(glutathione S-transferase) , PKC(protein kinase C) , Topo(DNAtopoisomerases), TK(tyrosine kinase), CD (bacterial cytosine deaminase), CPG2(carboxypeptidase G2) ,and PNP (purine nucleoside phosphorylase), have been known to bear close relations to cancer. Theirspecific expression and influence on the process of tumor initiation, promotion and progressionattract scientists to apply them as a biochemical marker of certain malignant tumor, a predictor ofresponse in cancer chemotherapy; to apply them to drug design, tumor prevention and as adjuvant toradiotherapy or surgery.

Combining cytochrome P-450 3A4 modulators and cyclosporine or everolimus in transplantation is successful

AIM: To describe the long term follow-up of kidney allograft recipients receiving ketoconazole with calcineurin inhibitors(CNI) alone or combined with everolimus. METHODS: This is an open-label, prospective observational clinical trial in low immunologic risk patients who, after signing an Institutional Review Board approved consent form, were included in one of two groups. The first one(n = 59) received everolimus(target blood level, 3-8 ng/m L) and the other(n = 114) azathioprine 2 mg/kg per day or mycophenolate mofetyl(MMF) 2 g/d. Both groups also received tapering steroids, the cytochrome P-450 3A4(CYP3A4) modulator, ketoconazole 50-100 mg/d, and cyclosporine with C0 targets in the everolimus group of 200-250 ng/mL in 1 mo, 100-125 ng/m L in 2 mo, and 50-65 ng/m L thereafter, and in the azathioprine or MMF group of 250-300 ng/mL in 1 mo, 200-250 ng/mL in 2 mo, 180-200 ng/m L until 3-6 mo, and 100-125 ng/mL thereafter. Clinical visits were performed monthly the first year and quarterly thereafter by treating physicians and all data was extracted by the investigators.RESULTS: The clinical characteristics of these two cohorts were similar. During the follow up(66 + 31 mo), both groups showed comparable clinical courses, but the biopsy proven acute rejection rate during the full follow-up period seemed to be lower in the everolimus group(20% vs 36%; P = 0.04). The everolimus group did not show a higher surgical complication rate thanthe other group. By the end of the follow-up period, the everolimus group tended to show a higher glomerular filtration rate. Nevertheless, we found no evidence of a consistent negative slope of the temporal allograft function estimated by the modification of the diet in renal disease formula in any of both groups. At 6 years of follow-up, the uncensored and death-censored graft survivals were 91% and 93%, and 91% and 83% in the everolimus plus cyclosporine, and cyclosporine alone groups, respectively. The addition of ketoconazole saved 80% of cyclosporine and 56% of everolimus doses. CONCLUSION: Combining CYP3A4 modulators with CNI or mammalian target of rapamycin inhibitor, in low immunological risk kidney transplant recipients is feasible, effective, safe and affordable even in the long term.

Studies on the Biomimetic Oxidation Catalyzed by the Model Compound of Cytochrome P-450 (Ⅶ) The Influence of the Axial Ligand X in TPPFe(Ⅲ)X on Its Catalytic Properties for the Oxygenation of Cyclohexane

In the study of cyclohexane monoxygenation with PhIO catalyzed by TPPFe( Ⅲ )X, we found the Influence of different axial ionic ligands X (X = F, Cl, Br, I, SCN, OR, R, CH,3, C2H6, (CH3)2CH, (CH2)3C)in TPPFe( Ⅲ )X on the oxidation products distribution and the yields of cyclohexanol. This paper deals with the linear relationship between the catalytic activity of TPPFe(Ⅲ)X and both the electronic or/ and steric effects of the axial ligands OR in TPPFe(Ⅲ)OR on its catalytic activity.

Cytochrome P450 2E1 genetic polymorphism and gastric cancer in Changle,Fujian Province

AIM: Genetic polymorphism in enzymes of carcinogen metabolism has been found to have the influence on the susceptibility to cancer. Cytochrome P450 2E1 (CYP2E1) is considered to play an important role in the metabolic activation of procarcinogens such as N-nitrosoamines and low molecular weight organic compounds. The purpose of this study is to determine whether CYP450 2E1 polymorphisms are associated with risks of gastric cancer. METHODS: We conducted a population based case-control study in Changle county, Fujian Province, a high-risk region of gastric cancer in China. Ninety-one incident gastric cancer patients and ninety-four healthy controls were included in our study. Datas including demographic characteristics, diet intake, and alcohol and tobacco consumption of individuals in our study were completed by a standardized questionnaire.PCR-RFLP revealed three genotypes:heterozygote (C1/C2) and two homozygotes (C1/C1 and C2/C2) in CYP2E1. RESULTS: The frequency of variant genotypes (C1/C2 and C2/C2) in gastric cancer cases and controls was 36.3% and 24.5%, respectively. The rare homozygous C2/C2 genotype was found in 6 individuals in gastric cancer group(6.6%), whereas there was only one in the control group (1.1%). However, there was no statistically significant difference between the two groups (two-tailed Fisher's exact test P=0.066). Individuals in gastric cancer group were more likely to carry genotype C1/C2 (odds ratio, OR=1.50) and C2/C2 (OR=7.34) than individuals in control group (chi(2) =4.597, for trend P=0.032). The frequencies of genotypes with the C2 allele (C1/C2 and C2/C2 genotypes) were compared with those of genotypes without C2 allele (C1/C1 genotype) among individuals in gastric cancer group and control group according to the pattern of gastric cancer risk factors. The results show that individuals who exposed to these gastric cancer risk factors and carry the C2 allele seemed to have a higher risk of developing gastric cancer. CONCLUSION: Polymorphism of CYP2E1 gene may have some effect in the development of gastric cancer in Changle county, Fujian Province.

Self-sufficient Cytochrome P450s and their potential applications in biotechnology

Cytochrome P450s(CYPs)are ubiquitously found in all kingdoms of life,playing important role in various biosynthetic pathways as well as degradative pathways;accordingly find applications in a vast variety of areas from organic synthesis and drug metabolite production to modification of biomaterials and bioremediation.Significantly,CYPs catalyze chemically challenging CAH and CAC activation reactions using a reactive high-valent iron-oxo intermediate generated upon dioxygen activation at their heme center,while the other oxygen atom is reduced to the level of water by electrons provided through a reductase partner protein.Self-sufficient CYPs,encoding their heme domain and reductase protein in a single polypeptide,facilitate increased catalytic efficiency and render a less complicated system to work with.The self-sufficient CYP enzyme from CYP102A family(CYP102A1,BM3)is among the earliest and most-investigated model enzymes for mechanistic and structural studies as well as for biotechnological applications.An increasing number of self-sufficient CYPs from the same CYP102 family and from other families have also been reported in last decade.In this review,we introduce chemistry and biology of CYPs,followed by an overview of the characteristics of self-sufficient CYPs and representative reactions.Enzyme engineering efforts leading to novel self-sufficient CYP variants that can catalyze synthetically useful natural and non-natural(nature-mimicking)reactions are highlighted.Lastly,the strategy and efforts that aim to circumvent the challenges for improved thermostability,regio-and enantioselectivity,and total turnover number;associated with practical use of self-sufficient CYPs are reviewed.

Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue

AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3, and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

Polymorphism of genes encoding drug-metabolizing and inflammation-related enzymes for susceptibility to cholangiocarcinoma in Thailand

BACKGROUND Cholangiocarcinoma(CCA)is an intractable cancer,and its incidence in north eastern Thailand is the highest worldwide.Infection with the liver fluke Opisthorchis viverrini(OV)has been associated with CCA risk.However,animal experiments have suggested that OV alone does not induce CCA,but its combination with a chemical carcinogen like nitrosamine can cause experimentally induced CCA in hamsters.Therefore,in humans,other environmental and genetic factors may also be involved.AIM To examine relations between risk for CCA and genetic polymorphisms in carcinogenmetabolizing and inflammation-related genes.METHODS This hospital-based case-control study enrolled 95 case-control pairs matched by age(±5 years)and sex.We examined relations between risk for CCA and genetic polymorphisms in carcinogenmetabolizing and inflammation-related genes,serum anti-OV,alcohol consumption,and smoking.Polymorphisms of CYP2E1,IL-6(-174 and-634),IL-10(-819),and NF-κB(-94)and their cooccurrence with polymorphisms in the drug-metabolizing enzyme gene GSTT1 or GSTM1 were also analyzed.RESULTS Although CCA risk was not significantly associated with any single polymorphism,persons with the GSTT1 wild-type and CYP2E1 c1/c2+c2/c2 genotype had an increased risk(OR=3.33,95%CI:1.23-9.00)as compared with persons having the GSTT1 wild-type and CYP2E1 c1/c1 wild genotype.The presence of anti-OV in serum was associated with a 7-to 11-fold increased risk,and smoking level was related to an OR of 1.5-1.8 in multivariable analyses adjusted for each of the seven genetic polymorphisms.CONCLUSION In addition to infection with OV,gene-gene interactions may be considered as one of the risk factors for CCA development.

Effects of Yanhusuo(Rhizoma Corydalis),Baizhi(Radix Angelicae Dahuricae)and Their Combination Extracts on Cytochrome P450 Activities in Rats

Background:Yuanhu Zhitong Prescription(元胡止痛方,YZP),a well-known herbal prescription for an analgesic effect,is recorded in the China Pharmacopoeia,consisting of Yanhusuo(Rhizoma Corydalis)and Baizhi(Radix Angelicae Dahuricae).Objective:To explore the influence of 70%EtOH extracts from Yanhusuo(Rhizoma Corydalis),Baizhi(Radix Angelicae Dahuricae)and YZP on the CYP450s,especially the differences between the single drug and prescription.Materials and methods:Cocktail probe drugs method was used to evaluate Cytochrome P450 activities in rat liver microsomes,including CYP1A2,CYP2D1,CYP2C11,CYP2C6 and CYP3A1,after rats repeatedly administrated with the extracts of Yanhusuo(Rhizoma Corydalis),Baizhi(Radix Angelicae Dahuricae)and YZP for 7 days.Results:Yanhusuo(Rhizoma Corydalis)extracts significantly increased the activities of CYP1A2,2C6 and 3A1,and inhibited that of 2D1.Baizhi(Radix Angelicae Dahuricae)extracts significantly increased the activities of CYP1A2 and inhibited that of 2D1 and 2C11.YZP extracts exhibited the same effect with single drugs.Conclusion:These results might partly interpret the TCM compatibility.Moreover,co-administration of prescriptions containing Yanhusuo(Rhizoma Corydalis),Baizhi(Radix Angelicae Dahuricae)or YZP should consider a potential herb(drug)-drug interaction medicated by the induction of CYP1A2,2C6 and 3A1 and inhibition of CYP2D1 and 2C11 enzymes.

丹参酮对大鼠细胞色素P-450酶系和谷胱甘肽转移酶的作用

目的探讨丹参有效成分丹参酮 (Tan)对大鼠肝、肾组织内的细胞色素P 450酶 (cytochromeP 450,CYP)系和谷胱甘肽转移酶(GT)的影响。方法给予SD雄性大鼠丹参酮 (100mg·kg-1 )灌胃,每日 1次,连续10天后,选用CYP1A1、1A2、2B1、2E1及 3A特异性代谢底物,检测它们在肝和肾微粒体中的活性,同时检测肝、肾微粒体中的GT水平变化。结果实验表明,丹参酮可诱导肝微粒体内的CYP1A1、CYP1A2和CYP2B1活性显著升高(均P<0. 01),同时显著抑制CYP2E1的活性 (P<0. 01),也可诱导肝微粒体中GT的活性升高 (P<0.05)。结论丹参酮对CYP亚型的不同调节作用可能会影响与其合用药物在体内的代谢消除,丹参酮对GT的影响也具有一定的临床意义。

A Bacterial Cytochrome P450 Enzyme Catalyzes Multistep Oxidation Reactions in Pyrroindomycin Biosynthesis

Cytochrome P450 enzymes (P450s) belong to a large family of oxidative hemeproteins and catalyze highly diverse oxygenation reactions that are involved in the biosynthesis of various natural products. Here, we report a multifunctional cytochrome P450 enzyme, PyrE2, which catalyzes the regioselective, successive 6-electron oxidation of an inert methyl group to produce a carboxyl product through formation of the hydroxyl and aldehyde intermediates in pyrroindomycin biosynthesis. The time-course biotransformation was characterized by the presence of the hydroxyl and aldehyde intermediates, the lag of the formation of the carboxyl product, and the subsequent loss of both intermediates, indicating that each 2-electron oxidation exhibits the distributive mechanism that requires substrate binding and product releasing. Bioinformatics analysis shows that the homologs of pyrE2 are common in the gene clusters of the spirotetronates varying in the oxidative state of the corresponding exocyclic carbon, indicating the generality and diversity of P450-catalyzed oxygenation in related biosynthetic pathways.

Engineering the Biosynthesis of Caffeic Acid in Saccharomyces cerevisiae with Heterologous Enzyme Combinations

Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two enzymes—HpaB and HpaC—from several bacteria, we constructed functional 4- hydroxyphenylacetate 3-hydroxylase (4HPA3H) in Saccharomyces cerevisiae to take on a role similar to that of the plant-derived cytochrome P450 enzyme and produce caffeic acid. Along with a common tyrosine ammonia lyase (TAL), the different combinations of HpaB and HpaC presented varied capabilities in producing the target product, caffeic acid, from the substrate, L-tyrosine. The highest production of caffeic acid was obtained with the enzyme combination of HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica, which yielded up to (289.4 ± 4.6) mg-L1 in shake-flask cultivation. The compatibility of heterologous enzymes within a yeast chassis was effectively improved, as the caffeic acid production was increased by 40 times from the initial yield. Six key amino acid residues around the flavin adenine dinucleotide (FAD) binding domain in HpaB from Pseudomonas aeruginosa were differentiate from those other HpaBs, and might play critical roles in affecting enzyme activity. We have thus established an effective approach to construct a highly efficient yeast system to synthesize non-native hydroxylated phenylpropanoids.

Effects of Tianma(Rhizoma Gastrodiae)and Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis)on cytochrome P450 enzyme activities in rats

OBJECTIVE:To investigate the efficacy of Tianma(Rhizoma Gastrodiae)and Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis)on cytochrome P450(CYP450)enzyme activities in rats.METHODS:A cocktail strategy was followed to evaluate the influence of Tianma(Rhizoma Gastrodiae)and Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis)on the activities of CYP450 isoforms(CYP1A2,CYP3A4,CYP2E1,CYP2C19,CYP2C9,CYP2D6),which were determined by changes in the pharmacokinetic parameters of six probe drugs,theophylline,dapsone,chlorzoxazone,omeprazole,tolbutamide and dextromethorphan.Study groups included,Control group(CG),Tianma(Rhizoma Gastrodiae)group(TM),Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis)group(GT)and Tianma Gouteng(Gastrodia Uncaria)group(TMGT).RESULTS:No significant differences between Tianma(Rhizoma Gastrodiae)and control groups were found.Compared with the control group,in the Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis)group both the AUC and t1/2 of dapsone and tolbutamide were reduced,whereas the CL(clearance rate)of dapsone and tolbutamide were increased.Compared with the control group,in the Tianma Gouteng group,the AUC and t1/2 of dapsone and tolbutamide were reduced,the CL of dapsone and tolbutamide were increased,and the AUC and t1/2 of chlorzoxazone were increased and the CL of chlorzoxazone was reduced.CONCLUSION:Tianma(Rhizoma Gastrodiae)has no significant effect on the six CYP450 subtypes.The activities of CYP3A4 and CYP2C9 were increased by Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis).The activities of CYP3A4 and CYP2C9 were increased,whereas the activity of CYP32E1 was reduced by combined Tianma(Rhizoma Gastrodiae)and Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis).

Tobacco smoking and its drug interactions with comedications involving CYP and UGT enzymes and nicotine

Tobacco smoking is a global public health threat causing several illnesses including cardiovascular disease(Myocardial infarction), cerebrovascular disease(Stroke), peripheral vascular disease(Claudication), chronic obstructive pulmonary disease, asthma, reduced female infertility, sexual dysfunction in men, different types of cancer and many other diseases. It has been estimated in 2015 that approximately 1.3 billion people smoke, around the globe. Use of medications among smokers is more common, nowadays. This review is aimed to identify the medications affected by smoking, involving Cytochrome P450(CYP)and uridine diphosphate-glucuronosyltransferases(UGTs) enzymes and Nicotine. Polycyclic aromatic hydrocarbons(PAHs) of tobacco smoke have been associated with the induction of CYP enzymes such as CYP1A1, CYP1A2 and possibly CYP2E1 and UGT enzymes. The drugs metabolized by CYP1A1,CYP1A2, CYP2E1 and UGT enzymes might be affected by tobacco smoking and the smokers taking medications metabolized by those enzymes, may need higher doses due to decreased plasma concentrations through enhanced induction by PAHs of tobacco smoke. The prescribers and the pharmacists are required to be aware of medications affected by tobacco smoking to prevent the toxicityassociated complications during smoking cessation.

Immunohistochemical localization of cytochrome P450 enzymes 2C and 4A in the normal rat brain

Objective To localize cytochrome P450 enzymes 4A and 2C in central nervous cells of normal male rats.Methods Eight drug/alcohol untreated normal male rats (150-200 g of body weight) were treated by the optimized perfusion technique, then brain tissues were postfixed, paraffin-embedded and cut into series sections, which were labeled by the improved strept-avidin-biotin complex DAB-nickel enhancer (SABC-DAB-Ni) immunohistochemistry and hematoxylin & eosin (H & E) stain techniques.Results The immunohistochemical results indicated that P450 2C-11 enzyme was localized in diverse numbers of neurons as well as some neuroglial cells, with focal or defuse distribution in many brain regions such as cerebrum, thalamus, olfactory bulb, hypothalamus, brain-stem, hippocampus, cerebellum, interpositus nucleus, caudate-putamen, and globus pallidus. In contrast, no positive findings of P450 4A-2, 3 and 8 enzymes were obtained in the same animals. With high magnification, 2C-11 protein was able to be roughly observed on the endoplasmic reticulum of the rat neurons.Conclusions P450 2C-11 protein, rather than P450 4A-2, 3 and 8, may be a candidate of brain P450 enzymes in the normal male rats.

Cytochrome P450 Enzyme-Copper Phosphate Hybrid Nano-Flowers with Superior Catalytic Performances for Selective Oxidation of Sulfides

Cytochrome P450 enzyme-copper phosphate hybrid materials with flower-like shape were prepared with a simple but efficient coprecipitation method.The growth process of the hybrid flowers can be divided into three successive steps：coordination/nucleation,growth,and further ripen.The concentration of enzymes in the mother liquor exerted great influence on the morphology and surface enzyme content of the nano-composites.The catalytic performance in the reaction of selective oxidation of sulfide to sulfoxide was also investigated.The hybrid flowers exhibited superior catalytic performance：satisfied thioanisole conversion and selectivity to methyl phenyl sulfoxide （both above 97%） with H2O2 as oxidant under mild reaction conditions,excellent stability and recyclability,and wide scope of substrates.Such results indicate that the hybrid materials are potentially good candidates in the industrial enzyme catalysis.

A practical strategy to develop isoform-selective near-infrared fluorescent probes for human cytochrome P450 enzymes

Currently,the development of selective fluorescent probes toward targeted enzymes is still a great challenge,due to the existence of numerous isoenzymes that share similar catalytic capacity.Herein,a double-filtering strategy was established to effectively develop isoenzyme-specific fluorescent probe(s)for cytochrome P450(CYP)which are key enzymes involving in metabolism of endogenous substances and drugs.In the first-stage of our filtering approach,near-infrared(NIR)fluorophores with alkoxyl group were prepared for the screening of CYP-activated fluorescent substrates using a CYPs-dependent incubation system.In the second stage of our filtering approach,these candidates were further screened using reverse protein-ligand docking to effectively determine CYP isoenzyme-specific probe(s).Using our double-filtering approach,probes S9 and S10 were successfully developed for the real-time and selective detection of CYP2C9 and CYP2J2,respectively,to facilitate high-throughput screening and assessment of CYP2C9-mediated clinical drug interaction risks and CYP2J2-associated disease diagnosis.These observations suggest that our strategy could be used to develop the isoform-specific probes for CYPs.

Functional expression and regulation of eukaryotic cytochrome P450 enzymes in surrogate microbial cell factories

Cytochrome P450(CYP)enzymes play crucial roles during the evolution and diversification of ancestral monocel-lular eukaryotes into multicellular eukaryotic organisms due to their essential functionalities including catalysis of housekeeping biochemical reactions,synthesis of diverse metabolites,detoxification of xenobiotics,and con-tribution to environmental adaptation.Eukaryotic CYPs with versatile functionalities are undeniably regarded as promising biocatalysts with great potential for biotechnological,pharmaceutical and chemical industry applica-tions.Nevertheless,the modes of action and the challenges associated with these membrane-bound proteins have hampered the effective utilization of eukaryotic CYPs in a broader range.This review is focused on comprehen-sive and consolidated approaches to address the core challenges in heterologous expression of membrane-bound eukaryotic CYPs in different surrogate microbial cell factories,aiming to provide key insights for better studies and applications of diverse eukaryotic CYPs in the future.We also highlight the functional significance of the previously underrated cytochrome P450 reductases(CPRs)and provide a rational justification on the progression of CPR from auxiliary redox partner to function modulator in CYP catalysis.