Abstract：
: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, >107 fold 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.
journal_name
ACS Chem Bioljournal_title
ACS chemical biologyauthors
Hoque MA,Zhang Y,Chen L,Yang G,Khatun MA,Chen H,Hao L,Feng Ydoi
10.1021/acschembio.7b00018subject
Has Abstractpub_date
2017-05-19 00:00:00pages
1188-1193issue
5eissn
1554-8929issn
1554-8937journal_volume
12pub_type
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