Structural Redesign of Lipase B from Candida antarctica by Circular Permutation and Incremental Truncation

JOURNAL OF MOLECULAR BIOLOGY

卷:393

Article

Qian, Zhen¹  Horton, John R.²  Cheng, Xiaodong²  Lutz, Stefan¹ 

[1] Emory Univ, Dept Chem, Atlanta, GA 30322 USA

[2] Emory Univ, Dept Biochem, Atlanta, GA 30322 USA

关键词: Candida antartica lipase B;    circular permutation;    enzyme engineering;    incremental truncation;   

DOI  :  10.1016/j.jmb.2009.08.008

来源: Elsevier

【 摘 要 】

Circular permutation of Candida antarctica lipase B yields several enzyme variants with substantially increased catalytic activity. To better understand the structural and functional consequences of protein termini reorganization, we have applied protein engineering and x-ray crystallography to cp283, one of the most active hydrolase variants. Our initial investigation has focused on the role of an extended surface loop, created by linking the native N- and C-termini, on protein integrity. Incremental truncation of the loop partially compensates for observed losses in secondary structure and the permutants' temperature of unfolding. Unexpectedly, the improvements are accompanied by quaternary-structure changes from monomer to dimer. The crystal structures of one truncated variant (cp283 Delta 7) in the apo-form determined at 1.49 angstrom resolution and with a bound phosphonate inhibitor at 1.69 angstrom resolution confirmed the formation of a homodimer by swapping of the enzyme's 35-residue N-terminal region. Separately, the new protein termini at amino acid positions 282/283 convert the narrow access tunnel to the catalytic triad into a broad crevice for accelerated substrate entry and product exit while preserving the native active-site topology for optimal catalytic turnover. (C) 2009 Elsevier Ltd. All rights reserved.

【 授权许可】

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