3u1o

THREE DIMENSIONAL STRUCTURE OF DE NOVO DESIGNED CYSTEINE ESTERASE ECH19, Northeast Structural Genomics Consortium Target OR49

spinqualitylabelsall models 3u1o, resolution 2.49Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3u1o is a 2 chain structure with sequence from Synthetic construct sequences. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: NA, SO4 NonStd Res: MSE Related: 2uvj Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity. Computational design of catalytic dyads and oxyanion holes for ester hydrolysis.,Richter F, Blomberg R, Khare SD, Kiss G, Kuzin AP, Smith AJ, Gallaher J, Pianowski Z, Helgeson RC, Grjasnow A, Xiao R, Seetharaman J, Su M, Vorobiev S, Lew S, Forouhar F, Kornhaber GJ, Hunt JF, Montelione GT, Tong L, Houk KN, Hilvert D, Baker D J Am Chem Soc. 2012 Oct 3;134(39):16197-206. doi: 10.1021/ja3037367. Epub 2012, Sep 21. PMID:22871159 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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