Design and evolution of new catalytic activity with an existing protein scaffold

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The design of enzymes with new functions and properties has long been a goal in protein engineering. Here, we report a strategy to change the catalytic activity of an existing protein scaffold. This was achieved by simultaneous incorporation and adjustment of functional elements through insertion, deletion, and substitution of several active site loops, followed by point mutations to fine-tune the activity. Using this approach, we were able to introduce beta-lactamase activity into the alpha beta/beta alpha metallohydrolase scaffold of glyoxalase II. The resulting enzyme, evMBL8 (evolved metallo beta-lactamase 8), completely lost its original activity and, instead, catalyzed the hydrolysis of cefotaxime with a (k(cat)/K-m)(app) of 1.8 x 10(2) (mole/titer)(-1) second(-1), thus increasing resistance to Escherichia coli growth on cefotaxime by a factor of about 100.
Publisher
AMER ASSOC ADVANCEMENT SCIENCE
Issue Date
2006-01
Language
English
Article Type
Article
Keywords

METALLO-BETA-LACTAMASE; BACTEROIDES-FRAGILIS; CRYSTAL-STRUCTURE; GLYOXALASE-II; BINDING; SITE; INHIBITOR; MECHANISM; COMPLEX; ENZYME

Citation

SCIENCE, v.311, no.5760, pp.535 - 538

ISSN
0036-8075
DOI
10.1126/science.1118953
URI
http://hdl.handle.net/10203/18150
Appears in Collection
CH-Journal Papers(저널논문)BS-Journal Papers(저널논문)
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