title: Trp266 determines the binding specificity of a porcine aflatoxin B₁ aldehyde reductase for aflatoxin B₁-dialdehyde.
abstract: Aflatoxin B₁ (AFB₁) is a severe threat to human and animal health. The aflatoxin B₁ aldehyde reductase (AFAR) family specifically catalyzes AFB₁-dialdehyde, a toxic metabolic intermediate of AFB₁, producing a nontoxic dialcohol. Although several AFARs have been found and characterized, the binding specificity of the family for AFB₁-dialdehyde remains unclear. Herein, according to the published sequence, we cloned a porcine AFAR gene. Recombinant porcine AFAR was expressed and purified from Escherichia coli as hexa-histidine tagged fusion protein. Using the cloned porcine AFAR as a model, site-directed mutagenesis combined with high performance liquid chromatography studies revealed that the substitution of Trp266 with Ala resulted in almost complete loss of catalytic activity for AFB₁-dialdehyde. Interestingly, the substitution of Met86 with Ala exhibited an obviously increased activity to the dialdehyde. Based on these results and by using molecular docking simulations, this work provides a structural explanation for why the AFAR family exhibits high specificity for AFB₁-dialdehyde. The Trp266 residue in porcine AFAR plays a critical role in stabilizing the binding of AFB₁-dialdehyde in the active pocket through the hydrophobic interaction of the side-chain indole ring of Trp266 with the fused coumarin rings of the dialdehyde molecule. The enhanced activity of M86A may be attributed to the formed π-π stacking interaction between Trp266 and the dialdehyde. In addition, other hydrophobic residues (e.g. Phe and Trp) around the dialdehyde molecule also stabilize the substrate binding. The findings may contribute to understanding the substrate specificity of the AFAR family for AFB₁-dialdehyde.
authors.affiliation: Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong 510642, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
authors.forename: Weiying
authors.initials: W
authors.lastname: Xu
authors.forename: Caihui
authors.initials: C
authors.lastname: Zhang
authors.forename: Qing
authors.initials: Q
authors.lastname: Chang
authors.forename: Xianqing
authors.initials: X
authors.lastname: Tang
authors.forename: Kangbai
authors.initials: K
authors.lastname: Li
authors.forename: Yiqun
authors.initials: Y
authors.lastname: Deng
mesh_terms: D016604:Aflatoxin B1 / Q000378:metabolism; D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D007700:Kinetics; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013552:Swine / Q000235:genetics; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014364:Tryptophan; D016604:Aflatoxin B1; C082765:aflatoxin B1 aldehyde reductase; D000449:Aldehyde Reductase
keywords: 2-CBA; 2-carboxybenzaldehyde; AFAR; AFB(1); AFB(1) exo-8,9-epoxide; AFB(1)-dialdehyde; AFBO; AFG(1); AKR; Aflatoxin B(1); Aflatoxin B(1) aldehyde reductase; CD; HPLC; Hydrophobic interaction; IPTG; Molecular docking; RMSD; SSA; Substrate specificity; WT; aflatoxin B(1); aflatoxin B(1) aldehyde reductase; aflatoxin G(1); aldo-keto reductase; circular dichroism; high performance liquid chromatography; isopropyl-β-d-thiogalactoside; root mean square deviation; succinic semialdehyde; wide-type