Regioselectivity Of Human CYP1A2 Towards Aflatoxin B₁

Aflatoxin is a kind of secondary metabolites produced by fungi,widely distributed in peanut,corn,soybean and other food crops.Aflatoxin B₁（Aflatoxin B₁,abbreviation AFB₁）is the most toxic of aflatoxin.AFB₁ in the liver was activated by cytochrome 450,transforming into a strong carcinogen,causing cancer.Cytochrome P450（cytochrome P450,abbreviated CYP）is a kind of important ultra-familial protein,which is extensively involved in the metabolism of various internal and external compounds in human and animal bodies.Human CYP1A2 is one of the key enzymes of metabolic AFB₁in the liver,and it is unclear to the molecular mechanism of human CYP1A2 combining AFB₁.According to the Escherichia coli preference codon,the whole C YP1A2 gene sequence was optimized,and the 17αsignaling peptide sequences were added to the 5’end of gene,the protein that was purified by N i²⁺affinity for the expression of DH5αin Escherichia coli was obtained by using pCWori+expression vector.CYP1A2 recombinant protein expression can reach 6 mg/L medium,its Fe²⁺·CO difference spectra have typical 450 nm absorption peaks.In order to construct the C YP enzyme reactive system,this study cloned human CYP450 reductase gene from human 293T cells,the 5-end introduced pelB signal peptide,and the expression of the active protein in BL21（DE3）PLysS was purified by pET22b,and the recombinant protein expressed in 20 mg/L medium was reached.Based on the CYP1A2 crystal structure of the reported person（PDB:2HI4）,using AutoDock 4.2 to simulate the molecular docking of AFB₁ and people CYP1A2,predict T124,F125,F226,F260 may participate in AFB₁ in the reactive pocket of the combination.Aiming at the target point mutation,expressing purification of mutants,using Fe²⁺·CO difference spectroscopy to identify the protein feature absorption peaks and determine the effective concentration.In order to obtain the AFB₁-GSH standard,we first use butyl hydroxy anise ether to induce the high expression of glutathione-S-transfer enzyme（GST）in mice liver,and purify the GST from rat hepatic cytoplasm.And then incubate t he AFB₁ with pig liver granules and add the GST binding AFBO of mice.HPLC collects AFB₁-GSH product peaks.The standard curves were established by HPLC after UV absorption spectrometry was determined by AFB₁-GSH concentration.Enzymatic incubation of CYP1A2 and mutant and AFB₁ was performed,and the rate of formation of AFB₁-GSH and AFM₁ products was quantitatively determined by HPLC.According to the standard curve,the CYP1A2metabolic AFB₁ produced AFBO V_max for 0.19 nmol/min/nmol CYP,K_m for 28μM,hydroxyl generation AFM₁ V_max for 0.60 nmol/min/nmol CYP,K_m to 22μM.The mutant F226A had no metabolic activity in AFB₁,and the mutant F226W and F226Y were more than 90%,but the activity was recovered compared to the mutant of F226A.The metabolic activity of mutant T124A to AFB₁ was not very different from the wild type,but the affinity of substrate was increased.The mutant F125A cannot be expressed normally;F260A mutants can be obtained,but the protein does not have the characteristic absorption p eaks of 450 nm.In order to further determine the specificity of the predicted amino acid sites,we examined the metabolic activity of recombinant human CYP1A2 to the AFB₁ of 7-ethoxyresorufin.Test to generate the V_max for 4.2 nmol/min/nmol CYP,K_m for 246 nm.The mutant F226A had no metabolic activity in the 7-ethoxyresorufin.At the same time,F226W,F226Y compared to wild type,metabolic activity decreased more than 84%,but relative to F226A mutant,the activity had some recovery.The activity o f mutant T124A to 7-ethoxyresorufin was not changed significantly in comparison with wild type.PyMO L analysis of molecular structure software shows that the benzene side chains of F226 and F260 may interact with AFB₁ molecules in sigma-πto maintain the union of AFB₁ active pockets.At the same time,because of AFB₁ and 7-ethoxyresorufin test halogen spirit are aromatic heterocyclic structure,F226 and other amino acids residues in7-ethoxyresorufin of the combination may play a similar role in AFB₁.To sum up,using molecular docking and site-direct mutation,we identify the key amino acids residues of Phe226 for human CYP1A2 and AFB₁,which are combined with theσ-πinteraction stabilization AFB₁ in C YP1A2 active pockets.