Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.

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 Biol

journal_title

ACS chemical biology

authors

Hoque MA,Zhang Y,Chen L,Yang G,Khatun MA,Chen H,Hao L,Feng Y

doi

10.1021/acschembio.7b00018

subject

Has Abstract

pub_date

2017-05-19 00:00:00

pages

1188-1193

issue

5

eissn

1554-8929

issn

1554-8937

journal_volume

12

pub_type

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