Structure Report for: 3ICW

Horton, J.R., Qian, Z., Jia, D., Lutz, S.A., Cheng, X. (3ICW replace 2R9D on 18 July 2009)

Title: Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation
Qian Z, Horton JR, Cheng X, Lutz S
Ref: Journal of Molecular Biology, 393:191, 2009 : PubMed
Abstract


ESTHER: Qian_2009_J.Mol.Biol_393_191
PubMedSearch: Qian 2009 J.Mol.Biol 393 191
PubMedID: 19683009
Gene_locus related to this paper: canar-LipB
Inhibitor(s) related to this paper: 4-methylumbelliferyl-hexylphosphonate

Abstract

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 A resolution and with a bound phosphonate inhibitor at 1.69 A 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.