8yfy | pdb_00008yfy

CRYSTAL STRUCTURE OF THE EST1 H274D MUTANT AT PH 4.2

spinqualitylabelsall models 8yfy, resolution 1.45Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 8yfy is a 1 chain structure with sequence from Saccharolobus shibatae. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.45Å Ligands: BOG Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function Q5NU42_9CREN Publication Abstract from PubMed SshEstI, a carboxylesterase from the thermoacidophilic archaeon Saccharolobus shibatae, is a member of the hormone-sensitive lipase family that displays slightly alkaliphilic activity with an optimum activity at pH 8.0. In this study, three distinct strategies were explored to confer acidophilic properties to SshEstI. The first strategy involved engineering the oxyanion hole by replacing Gly81 with serine or aspartic acid. The G81S mutant showed optimum activity at pH 7.0, whereas the aspartic acid mutant (G81D) rendered the enzyme slightly acidophilic with optimum activity observed at pH 6.0; however, k(cat) and k(cat)/K(m) values were reduced by these substitutions. The second strategy involved examining the effects of surfactant additives on the pH-activity profiles of SshEstI. The results showed that cetyltrimethylammonium bromide (CTAB) enhanced wild-type enzyme (WT) activity at acidic pH values. In the presence of 0.1 mM CTAB, G81S and G81D were acidophilic enzymes with optimum activity at pH 6.0 and 4.0, respectively, although their enzyme activities were low. The third strategy involved engineering the active site to resemble that of kumamolisin-As (kuma-As), an acidophilic peptidase of the sedolisin family. The catalytic triad of kuma-As was exchanged into SshEstI using site-directed mutagenesis. X-ray crystallographic analysis of the mutants (H274D and H274E) revealed that the potential hydrogen donor-acceptor distances around the active site of WT were fully maintained in these mutants. However, these mutants were inactive at pH 4-8. Lowering pH optimum of activity of SshEstI, a slightly alkaliphilic archaeal esterase of the hormone-sensitive lipase family.,Ohara K, Oshima Y, Unno H, Nagano S, Kusunoki M, Takahashi S, Waki T, Yamashita S, Nakayama T J Biosci Bioeng. 2024 Jun 24:S1389-1723(24)00158-0. doi: , 10.1016/j.jbiosc.2024.05.010. PMID:38918133 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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