Key residues for controlling enantioselectivity of Halohydrin dehalogenase from Arthrobacter sp. strain AD2, revealed by structure-guided directed evolution.
Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a valuable tool in the preparation of R enantiomers of epoxides and β-substituted alcohols. In contrast, the halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) shows a low S enantioselectivity toward most aromatic substrates. Here, three amino acids (V136, L141, and N178) located in the two neighboring active-site loops of HheA were proposed to be the key residues for controlling enantioselectivity. They were subjected to saturation mutagenesis aimed at evolving an S-selective enzyme. This led to the selection of two outstanding mutants (the V136Y/L141G and N178A mutants). The double mutant displayed an inverted enantioselectivity (from S enantioselectivity [E(S)] = 1.7 to R enantioselectivity [E(R)] = 13) toward 2-chloro-1-phenylethanol without compromising enzyme activity. Strikingly, the N178A mutant showed a large enantioselectivity improvement (E(S) > 200) and a 5- to 6-fold-enhanced specific activity toward (S)-2-chloro-1-phenylethanol. Further analysis revealed that those mutations produced some interference for the binding of nonfavored enantiomers which could account for the observed enantioselectivities. Our work demonstrated that those three active-site residues are indeed crucial in modulating the enantioselectivity of HheA and that a semirational design strategy has great potential for rapid creation of novel industrial biocatalysts.
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China. lixiatang@uestc.edu.cn
Lixia
L
Tang
Xuechen
X
Zhu
Huayu
H
Zheng
Rongxiang
R
Jiang
Maja
M
Majeric Elenkov
D019943:Amino Acid Substitution; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D019020:Directed Molecular Evolution; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D050505:Mutant Proteins; D006867:Hydrolases; C430848:halohydrin dehalogenase