Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation.
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.
- Research Organization:
- Argonne National Lab. (ANL), Argonne, IL (United States)
- Sponsoring Organization:
- USDOE
- OSTI ID:
- 1005948
- Journal Information:
- J. Mol. Biol., Vol. 393, Issue (1) ; 10, 2009; ISSN 0022-2836
- Country of Publication:
- United States
- Language:
- ENGLISH
Similar Records
Effects of Porous Polystyrene Resin Parameters on Candida antarctica Lipase B Adsorption, Distribution, and Polyester Synthesis Activity
Effects of Macroporous Resin Size on Candida antarctica Lipase B Adsorption, Fraction of Active Molecules, and Catalytic Activity for Polyester Synthesis