Design and evolution of new catalytic activity with an existing protein scaffold.
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.
Park
Hee-Sung
HS
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-Dong, Yusung-Gu, Daejon 305-701, Korea.
Nam
Sung-Hun
SH
Lee
Jin Kak
JK
Yoon
Chang No
CN
Mannervik
Bengt
B
Benkovic
Stephen J
SJ
Kim
Hak-Sung
HS
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002439:Cefotaxime / Q000378:metabolism / Q000494:pharmacology; D019020:Directed Molecular Evolution; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects; D019143:Evolution, Molecular; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*; D000099301:Hydroxyacylglutathione Hydrolase
D002439:Cefotaxime; D007501:Iron; D008670:Metals; D011993:Recombinant Fusion Proteins; D013869:Thiolester Hydrolases; D015032:Zinc; D001618:beta-Lactamases; D000099301:Hydroxyacylglutathione Hydrolase; C409201:beta-lactamase IMP-1