Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.
The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >107fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai 200240, China.
Md Anarul
MA
Hoque
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai 200240, China.
Yong
Y
Zhang
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai 200240, China.
Liuqing
0000-0002-2104-3697
L
Chen
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai 200240, China.
Guangyu
G
Yang
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai 200240, China.
Mst Afroza
MA
Khatun
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai 200240, China.
Haifeng
H
Chen
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai 200240, China.
Liu
L
Hao
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai 200240, China.
Yan
0000-0002-2522-2115
Y
Feng
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics*; D056506:Geobacillus / Q000201:enzymology*; D062105:Molecular Docking Simulation; D015394:Molecular Structure; D016254:Mutagenesis, Insertional / Q000379:methods*; D010755:Organophosphates / Q000378:metabolism; D044345:Phosphoric Triester Hydrolases / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010755:Organophosphates; D002265:Carboxylic Ester Hydrolases; D044345:Phosphoric Triester Hydrolases