| Title : Morphing activity between structurally similar enzymes: from heme-free bromoperoxidase to lipase - Chen_2009_Biochemistry_48_11496 |
| Author(s) : Chen B , Cai Z , Wu W , Huang Y , Pleiss J , Lin Z |
| Ref : Biochemistry , 48 :11496 , 2009 |
| Abstract : In this study, to explore the plasticity of the alpha/beta-hydrolase fold family, we converted bromoperoxidase A2 (BPO-A2) from Streptomyces aureofaciens to a lipase by structure comparison with lipase A (LipA) from Bacillus subtilis. These two enzymes have similar structures (2.1 A rmsd) and a very low level of sequence identity ( approximately 18%). A variant BL1 was constructed by deleting the caplike domain of BPO-A2 and further fine-tuning the newly formed substrate binding site. The lipase activity was successfully transplanted on BL1, while the halogenation activity was totally lost. BL1 also showed higher hydrolytic activities toward long chain p-nitrophenyl esters, such as p-nitrophenyl caprylate (3.7-fold) and p-nitrophenyl palmitate (7.0-fold), while its activity toward a short chain ester (p-nitrophenyl acetate) decreased dramatically, to only 1.2% of that of BPO-A2. After two rounds of directed evolution and site-directed mutagenesis on selected residues, several mutants with both improved hydrolytic activities and substrate preferences toward long chain substrates were obtained. The highest hydrolytic activity toward p-nitrophenyl palmitate of the best mutant BL1-2-E8-plusI was improved by 40-fold compared with that of BL1. These results demonstrate the possibility of manipulating the caplike domain of alpha/beta-hydrolase fold enzymes and provide further understanding of the structure-function relationship of the alpha/beta-hydrolase fold enzymes. The design strategy used in this study could serve as a useful approach for constructing variants with targeted catalytic properties using the alpha/beta-hydrolase fold. |
| ESTHER : Chen_2009_Biochemistry_48_11496 |
| PubMedSearch : Chen_2009_Biochemistry_48_11496 |
| PubMedID: 19883129 |
Chen B, Cai Z, Wu W, Huang Y, Pleiss J, Lin Z (2009)
Morphing activity between structurally similar enzymes: from heme-free bromoperoxidase to lipase
Biochemistry
48 :11496
Chen B, Cai Z, Wu W, Huang Y, Pleiss J, Lin Z (2009)
Biochemistry
48 :11496