title: The "gate keeper" role of Trp222 determines the enantiopreference of diketoreductase toward 2-chloro-1-phenylethanone.
abstract: Trp222 of diketoreductase (DKR), an enzyme responsible for reducing a variety of ketones to chiral alcohols, is located at the hydrophobic dimeric interface of the C-terminus. Single substitutions at DKR Trp222 with either canonical (Val, Leu, Met, Phe and Tyr) or unnatural amino acids (UAAs) (4-cyano-L-phenylalanine, 4-methoxy-L-phenylalanine, 4-phenyl-L-phenyalanine, O-tert-butyl-L-tyrosine) inverts the enantiotope preference of the enzyme toward 2-chloro-1-phenylethanone with close side chain correlation. Analyses of enzyme activity, substrate affinity and ternary structure of the mutants revealed that substitution at Trp222 causes a notable change in the overall enzyme structure, and specifically in the entrance tunnel to the active center. The size of residue 222 in DKR is vital to its enantiotope preference. Trp222 serves as a "gate keeper" to control the direction of substrate entry into the active center. Consequently, opposite substrate-binding orientations produce respective alcohol enantiomers.
authors: State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, Jiangsu Province, People's Republic of China.
authors: Hairong
authors: H
authors: Ma
authors: State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, Jiangsu Province, People's Republic of China.
authors: Xin
authors: X
authors: Yang
authors: State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, Jiangsu Province, People's Republic of China.
authors: Zhuo
authors: Z
authors: Lu
authors: State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, Jiangsu Province, People's Republic of China.
authors: Nan
authors: N
authors: Liu
authors: State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, Jiangsu Province, People's Republic of China.
authors: Yijun
authors: Y
authors: Chen
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013237:Stereoisomerism; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism; D002721:omega-Chloroacetophenone / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D002721:omega-Chloroacetophenone; D014364:Tryptophan; D010088:Oxidoreductases