Park, HS Nam, SH Lee, JK Yoon, CN Mannervik, B Benkovic, SJ Kim, HS 2024-01-21T03:42:39Z 2024-01-21T03:42:39Z 2021-09-02 2006-01 0036-8075 https://pubs.kist.re.kr/handle/201004/135846 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. English AMER ASSOC ADVANCEMENT SCIENCE Design and evolution of new catalytic activity with an existing protein scaffold Article 10.1126/science.1118953 1 SCIENCE, v.311, no.5760, pp.535 - 538 SCIENCE 311 5760 535 538 N scie scopus 000235071400049 2-s2.0-31544477181 Multidisciplinary Sciences Science & Technology - Other Topics Article METALLO-BETA-LACTAMASE BACTEROIDES-FRAGILIS CRYSTAL-STRUCTURE GLYOXALASE-II BINDING SITE INHIBITOR MECHANISM COMPLEX ENZYME protein design scaffold evolution