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 (cp283Δ7) in the apo-form determined at 1.49Å resolution and with a bound phosphonate inhibitor at 1.69Å 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.
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