4r9l

Structure of a thermostable elevenfold mutant of limonene epoxide hydrolase from Rhodococcus erythropolis, containing two stabilizing disulfide bonds

spinqualitylabelsall models 4r9l, resolution 1.80Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 4r9l is a 3 chain structure with sequence from Rhodococcus erythropolis. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.8Å Ligands: HYH Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function LIMA_RHOER Catalyzes the conversion of limonene-1,2-epoxide to limonene-1,2-diol. Can use both the (-) and (+) isomers of limonene-1,2-epoxide as substrates and also has some activity with 1-methylcyclohexene oxide, cyclohexene oxide and indene oxide as substrates. Publication Abstract from PubMed Protein engineering aimed at enhancing enzyme stability is increasingly supported by computational methods for calculation of mutant folding energies and for the design of disulfide bonds. To examine the accuracy of mutant structure predictions underlying these computational methods, crystal structures of thermostable limonene epoxide hydrolase variants obtained by computational library design were determined. Four different predicted effects indeed contributed to the obtained stabilization: (i) enhanced interactions between a flexible loop close to the N-terminus and the rest of the protein; (ii) improved interactions at the dimer interface; (iii) removal of unsatisfied hydrogen bonding groups; and (iv) introduction of additional positively charged groups at the surface. The structures of an eightfold and an elevenfold mutant showed that most mutations introduced the intended stabilizing interactions, and side-chain conformations were correctly predicted for 72-88% of the point mutations. However, mutations that introduced a disulfide bond in a flexible region had a larger influence on the backbone conformation than predicted. The enzyme active sites were unaltered, in agreement with the observed preservation of catalytic activities. The structures also revealed how a c-Myc tag, which was introduced for facile detection and purification, can reduce access to the active site and thereby lower the catalytic activity. Finally, sequence analysis showed that comprehensive mutant energy calculations discovered stabilizing mutations that are not proposed by the consensus or B-FIT methods. This article is protected by copyright. All rights reserved. X-ray crystallographic validation of structure predictions used in computational design for protein stabilization.,Floor RJ, Wijma HJ, Jekel PA, Terwisscha van Scheltinga AC, Dijkstra BW, Janssen DB Proteins. 2015 Mar 4. doi: 10.1002/prot.24791. PMID:25739581 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Epoxide hydrolase 3D structures

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