Alteration of substrate specificities of thermophilic α/βhydrolases through domain swapping and domain interface optimization  

作　　者：Xiaoli Zhou Honglei Wang Yuhang Zhang Le Gao Yan Feng 

机构地区：[1]State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China [2]Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, China

出　　处：《Acta Biochimica et Biophysica Sinica》2012年第12期965-973,共9页生物化学与生物物理学报(英文版)

摘　　要：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 α/β hydrolase superfamily by grafting their functional domains to the conserved α/β hydrolase fold domain: a earboxylesterase from Archaeoglobusfulgidus (AFEST) and an acylpeptide hydrolase from Aeropyrum pernix K1 (apAPH) and explored their activities on hydrolyze p-nitrophenyl esters (pNP) with different acyi chain lengths. We took two approaches to reduce the crossover disruptions when cre- ating 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 specifi- city, 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 sub- strate-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.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 α/β hydrolase superfamily by grafting their functional domains to the conserved α/β hydrolase fold domain: a earboxylesterase from Archaeoglobusfulgidus (AFEST) and an acylpeptide hydrolase from Aeropyrum pernix K1 (apAPH) and explored their activities on hydrolyze p-nitrophenyl esters (pNP) with different acyi chain lengths. We took two approaches to reduce the crossover disruptions when cre- ating 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 specifi- city, 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 sub- strate-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.

关 键 词：domain swapping thermophilic esterase α/βhydrolase superfamily 

分 类 号：Q556[生物学—生物化学] TS202.3[轻工技术与工程—食品科学]