Many lipases are potent catalysts of stereoselective reactions and are therefore of interest for use in chemical synthesis. The crystal structures of lipases show a large variation in the shapes of their active site environments that may explain the large variation in substrate specificity of these enzymes. We have determined the three-dimensional structure of Candida antarctica lipase B (CALB) cocrystallized with the detergent Tween 80. In another crystal form, the structure of the enzyme in complex with a covalently bound phosphonate inhibitor has been determined. In both structures, the active site is exposed to the external solvent. The potential lid-forming helix alpha 5 in CALB is well-ordered in the Tween 80 structure and disordered in the inhibitor complex. The tetrahedral intermediates of two chiral substrates have been modeled on the basis of available structural and biochemical information. The results of this study provide a structural explanation for the high stereoselectivity of CALB toward many secondary alcohols.
The peroxisome proliferator-activated receptors (PPAR) are members of the nuclear receptor supergene family and are considered as key sensors of both lipid and glucose homeostasis. The role of the PPAR␥ isoform in glucose metabolism is illustrated by the fact that antidiabetic thiazolidinediones have been shown to be bona fide PPAR␥ ligands. Here we report the crystal structure of apo-PPAR␥ ligand binding domain (LBD) determined to 2.9-Å resolution. Although the structure of apo-PPAR␥-LBD retains the overall fold described previously for other nuclear receptor LBDs, three distinct structural differences are evident. 1) The core AF-2 activation domain of apo-PPAR␥ LBD is folded back toward the predicted ligand binding pocket similar to that observed in the holo-forms of other nuclear receptors. 2) The proposed ligand binding pocket of apo-PPAR␥-LBD is larger and more accessible to the surface in contrast to other LBDs. 3) The region of the LBD called the -loop is extended in PPAR␥ and contains additional structural elements. Taken together, the apo-PPAR␥-LBD structure is in several aspects different from previously described LBDs. Given the central role of PPAR␥ as a mediator in glucose regulation, the structure should be an important tool in the development of improved anti-diabetic agents.
These observations strongly suggest that maltose phosphorylase has evolved from glucoamylase. MP has probably conserved one carboxylate group for acid catalysis and has exchanged the catalytic base for a phosphate binding pocket. The relative positions of the acid catalytic group and the bound phosphate are compatible with a direct-attack mechanism of a glycosidic bond by phosphate, in accordance with inversion of configuration at the anomeric carbon as observed for this enzyme.
The mammalian SPRY domain- and SOCS box-containing proteins, SPSB1 to SPSB4, belong to the SOCS box family of E3 ubiquitin ligases. Substrate recognition sites for the SPRY domain are identified only for human Par-4 (ELNNNL) and for the Drosophila orthologue GUSTAVUS binding to the DEAD-box RNA helicase VASA (DINNNN). To further investigate this consensus motif, we determined the crystal structures of SPSB1, SPSB2, and SPSB4, as well as their binding modes and affinities for both Par-4 and VASA. Mutation of each of the three Asn residues in Par-4 abrogated binding to all three SPSB proteins, while changing EL to DI enhanced binding. By comparison to SPSB1 and SPSB4, the more divergent protein SPSB2 showed only weak binding to Par-4 and was hypersensitive to DI substitution. Par-4(59–77) binding perturbed NMR resonances from a number of SPSB2 residues flanking the ELNNN binding site, including loop D, which binds the EL/DI sequence. Although interactions with the consensus peptide motif were conserved in all structures, flanking sites in SPSB2 were identified as sites of structural change. These structural changes limit high-affinity interactions for SPSB2 to aspartate-containing sequences, whereas SPSB1 and SPSB4 bind strongly to both Par-4 and VASA peptides.
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