role of the nc-loop in catalytic activity and stability in lipase from fervidobacterium changbaicumnc-loop角色在脂肪酶催化活性和稳定性从fervidobacterium changbaicum.pdfVIP

Role of the NC-Loop in Catalytic Activity and Stability in Lipase from Fervidobacterium changbaicum Binchun Li1,2., Guangyu Yang1., Lie Wu1,2, Yan Feng1,2* 1 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, P. R. China, 2 Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, P. R. China Abstract Flexible NC-loops between the catalytic domain and the cap domain of the a/ b hydrolase fold enzymes show remarkable diversity in length, sequence, and configuration. Recent investigations have suggested that the NC-loop might be involved in catalysis and substrate recognition in many enzymes from the a/ b hydrolase fold superfamily. To foster a deep understanding of its role in catalysis, stability, and divergent evolution, we here systemically investigated the function of the NC-loop (residues 131–151) in a lipase (FClip1) from thermophilic bacterium Fervidobacterium changbaicum by loop deletion, alanine-scanning mutagenesis and site-directed mutagenesis. We found that the upper part of the NC-loop (residues 131–138) was of great importance to enzyme catalysis. Single substitutions in this region could fine-tune the activity of FClip1 as much as 41-fold, and any deletions from this region rendered the enzyme completely inactive. The lower part of the NC-loop (residues 139–151) was capable of enduring extensive deletions without loss of activity. The shortened mutants in this region were found to show both improved activity and increased stability simultaneously. We therefore speculated that the NC-loop, especially the lower part, would be a perfect target for enzyme engineering to optimize the enzymatic prop