| Title : Alteration of substrate specificities of thermophilic alpha\/beta hydrolases through domain swapping and domain interface optimization - Zhou_2012_Acta.Biochim.Biophys.Sin.(Shanghai)_44_965 |
| Author(s) : Zhou X , Wang H , Zhang Y , Gao L , Feng Y |
| Ref : Acta Biochim Biophys Sin (Shanghai) , 44 :965 , 2012 |
| Abstract : Protein domain swapping is an efficient way in protein functional evolution in vivo and also has been proved to be an effective strategy to modify the function of the multi-domain proteins in vitro. To explore the potentials of domain swapping for alteration of the enzyme substrate specificities and the structure-function relationship of the homologous proteins, here we constructed two chimeras from a pair of thermophilic members of the alpha/beta hydrolase superfamily by grafting their functional domains to the conserved alpha/beta hydrolase fold domain: a carboxylesterase from Archaeoglobus fulgidus (AFEST) and an acylpeptide hydrolase from Aeropyrum pernix K1 (apAPH) and explored their activities on hydrolyze p-nitrophenyl esters (pNP) with different acyl chain lengths. We took two approaches to reduce the crossover disruptions when creating the chimeras: chose the residue which involved in the least contacts as the splicing site and optimized the newly formed domain interfaces of the chimeras by site-directed mutations. Characterizations of AAM7 and PAR showed that these chimeras inherited the thermophilic property of both parents. In the aspect of substrate specificity, AAM7 and PAR showed highest activity towards short chain length substrate pNPC4 and middle chain length substrate pNPC8, similar to parent AFEST and apAPH, respectively. These results suggested that the substrate-binding domain is the dominant factor on enzyme substrate specificity, and the optimization of the newly formed domain interface is an important guarantee for successful domain swapping of proteins with low-sequence homology. |
| ESTHER : Zhou_2012_Acta.Biochim.Biophys.Sin.(Shanghai)_44_965 |
| PubMedSearch : Zhou_2012_Acta.Biochim.Biophys.Sin.(Shanghai)_44_965 |
| PubMedID: 23099882 |
Zhou X, Wang H, Zhang Y, Gao L, Feng Y (2012)
Alteration of substrate specificities of thermophilic alpha\/beta hydrolases through domain swapping and domain interface optimization
Acta Biochim Biophys Sin (Shanghai)
44 :965
Zhou X, Wang H, Zhang Y, Gao L, Feng Y (2012)
Acta Biochim Biophys Sin (Shanghai)
44 :965