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Design and evolution of new catalytic activity with an existing protein scaffold.
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2006 (English)In: Science, ISSN 1095-9203, Vol. 311, no 5760, 535-8 p.Article in journal (Refereed) Published
Abstract [en]

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 alphabeta/betaalpha 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 (kcat/Km)app of 1.8 x 10(2) (mole/liter)(-1) second(-1), thus increasing resistance to Escherichia coli growth on cefotaxime by a factor of about 100.

Place, publisher, year, edition, pages
2006. Vol. 311, no 5760, 535-8 p.
Keyword [en]
Amino Acid Sequence, Binding Sites, Catalysis, Catalytic Domain, Cefotaxime/metabolism/pharmacology, Directed Molecular Evolution, Drug Resistance; Bacterial, Escherichia coli/drug effects, Evolution; Molecular, Humans, Hydrophobicity, Iron/metabolism, Kinetics, Metals/metabolism, Models; Molecular, Molecular Sequence Data, Point Mutation, Protein Conformation, Protein Engineering, Protein Folding, Protein Structure; Secondary, Protein Structure; Tertiary, Recombinant Fusion Proteins/metabolism, Research Support; Non-U.S. Gov't, Substrate Specificity, Thiolester Hydrolases/*chemistry/genetics/*metabolism, Zinc/metabolism, beta-Lactamases/chemistry/*metabolism
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Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-76736PubMedID: 16439663OAI: oai:DiVA.org:uu-76736DiVA: diva2:104648
Available from: 2006-03-07 Created: 2006-03-07 Last updated: 2011-01-11

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Mannervik, Bengt

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