Functional roles of Tryptophan residues in diketoreductase from Acinetobacter baylyi.
Diketoreductase (DKR) from Acinetobacter baylyi contains two tryptophan residues at positions 149 and 222. Trp-149 and Trp-222 are located along the entry path of substrate into active site and at the dimer interface of DKR, respectively. Single and double substitutions of these positions were generated to probe the roles of tryptophan residues. After replacing Trp with Ala and Phe, biochemical and biophysical characteristics of the mutants were thoroughly investigated. Enzyme activity and substrate binding affinity of W149A and W149F were remarkably decreased, suggesting that Trp-149 regulates the position of substrate at the binding site. Meanwhile, enzyme activity of W222F was increased by 1.7-fold while W222A was completely inactive. In addition to lower thermostability of Trp-222 mutants, molecular modeling of the mutants revealed that Trp-222 is vital to protein folding and dimerization of the enzyme.
State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, Jiangsu Province, China.
Yan
Y
Huang
Zhuo
Z
Lu
Min
M
Ma
Nan
N
Liu
Yijun
Y
Chen
D000150:Acinetobacter / Q000201:enzymology*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D019281:Dimerization; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014364:Tryptophan / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014364:Tryptophan; D009247:NADH, NADPH Oxidoreductases