Publikationsdatum:
2006-01-28
Beschreibung:
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.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Park, Hee-Sung -- Nam, Sung-Hun -- Lee, Jin Kak -- Yoon, Chang No -- Mannervik, Bengt -- Benkovic, Stephen J -- Kim, Hak-Sung -- New York, N.Y. -- Science. 2006 Jan 27;311(5760):535-8.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-Dong, Yusung-Gu, Daejon 305-701, Korea.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/16439663" target="_blank"〉PubMed〈/a〉
Schlagwort(e):
Amino Acid Sequence
;
Binding Sites
;
Catalysis
;
Catalytic Domain
;
Cefotaxime/metabolism/pharmacology
;
*Directed Molecular Evolution
;
Drug Resistance, Bacterial
;
Escherichia coli/drug effects
;
Evolution, Molecular
;
Humans
;
Hydrophobic and Hydrophilic Interactions
;
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
;
Substrate Specificity
;
Thiolester Hydrolases/*chemistry/genetics/*metabolism
;
Zinc/metabolism
;
beta-Lactamases/chemistry/*metabolism
Print ISSN:
0036-8075
Digitale ISSN:
1095-9203
Thema:
Biologie
,
Chemie und Pharmazie
,
Informatik
,
Medizin
,
Allgemeine Naturwissenschaft
,
Physik
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