title: Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation.
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.
authors.affiliation: Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Qian
authors.forename: John R
authors.initials: JR
authors.lastname: Horton
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Cheng
authors.forename: Stefan
authors.initials: S
authors.lastname: Lutz
mesh_terms: D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology*; D018360:Crystallography, X-Ray; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D008049:Lipase
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