Structure of Bovine Pancreatic Cholesterol Esterase at 1.6 Å:  Novel Structural Features Involved in Lipase Activation^,

Abstract

The structure of pancreatic cholesterol esterase, an enzyme that hydrolyzes a wide variety of dietary lipids, mediates the absorption of cholesterol esters, and is dependent on bile salts for optimal activity, is determined to 1.6 A resolution. A full-length construct, mutated to eliminate two N-linked glycosylation sites (N187Q/N361Q), was expressed in HEK 293 cells. Enzymatic activity assays show that the purified, recombinant, mutant enzyme has activity identical to that of the native, glycosylated enzyme purified from bovine pancreas. The mutant enzyme is monomeric and exhibits improved homogeneity which aided in the growth of well-diffracting crystals. Crystals of the mutant enzyme grew in space group C2, with the following cell dimensions: a = 100.42 A, b = 54.25 A, c = 106.34 A, and beta = 104.12 degrees, with a monomer in the asymmetric unit. The high-resolution crystal structure of bovine pancreatic cholesterol esterase (Rcryst = 21.1%; Rfree = 25.0% to 1.6 A resolution) shows an alpha-beta hydrolase fold with an unusual active site environment around the catalytic triad. The hydrophobic C terminus of the protein is lodged in the active site, diverting the oxyanion hole away from the productive binding site and the catalytic Ser194. The amphipathic, helical lid found in other triglyceride lipases is truncated in the structure of cholesterol esterase and therefore is not a salient feature of activation of this lipase. These two structural features, along with the bile salt-dependent activity of the enzyme, implicate a new mode of lipase activation.

Purification and Characterization of a Novel Cholesterol Esterase fromPseudomonas aeruginosa, with Its Application to Cleaning Lipid-stained Contact Lenses

“…28) Recently three-dimensional structures of bovine pancreatic cholesterol esterase and its complex with taurocholate have been elucidated by X-ray analysis. [29][30][31] Two taurocholate binding sites were found in the complex structure. One of these sites was close to the cholesterol esterase-speciˆc hairpin loop near the active site.…”

Carboxyl ester lipase

“…A truncated bovine CEL lacking the proline-rich repeating units at the carboxy-terminus was shown at 0.28 nm resolution to be a protein with 13 ␤ -strands and 14 ␣ -helices (20). However, a full-length bovine CEL structure resolved at 0.16 nm resolution indicated a protein with 11 ␤ -strands and 15 ␣ -helices (21). The X-ray crystal structure of a truncated human CEL showed that this protein is built around a core comprised of strongly twisted 11-stranded ␤ -sheets, with a smaller three-stranded ␤ -sheet found at the amino-terminus (22).…”

Proteins

“…While cutinase is the smallest enzyme of the a/β hydrolases, with five strands in the main β-sheet, 7 bovine bile-salt activated cholesterol esterase has 11 strands and loop structures up to 79 residues in length. 8 Divergence. There are essentially two types of protein structural divergence: changes to the proteins surface or peripheral regions (e.g., surface loops, surfaces helices and strands on the edges of β-sheets) and the less common but far more detrimental modifications to the proteins interior or core.…”