3h56

Met150Leu/Phe312Cys variant of nitrite reductase from Alcaligenes faecalis

spinqualitylabelsall models 3h56, resolution 1.50Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3h56 is a 1 chain structure with sequence from Alcaligenes faecalis. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.5Å Ligands: CSD, CU Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function NIR_ALCFA Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed Directed evolution methods were developed for Cu-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6. The PCR cloning strategy allows for the efficient production of libraries of 100 000 clones by a modification of a megaprimer-based whole-plasmid synthesis reaction. The high-throughput screen includes colony lift onto a nylon membrane and subsequent lysis of NiR-expressing colonies in the presence of Cu(2+) ions for copper incorporation into intracellularly expressed NiR. Addition of a chromogenic substrate, 3, 3'-diaminobenzidine (DAB), results in deposition of red, insoluble color at the site of oxidation by functional NiR. Twenty-thousand random variants of NiR were screened for improved function with DAB as a reductant, and five variants were identified. These variants were shuffled and screened, yielding two double variants. An analog of the DAB substrate, o-dianisidine, which is oxidized to a water-soluble product was used for functional characterization. The double variant M150L/F312C was most proficient at o-dianisidine oxidation with dioxygen as the electron acceptor (5.5X wt), and the M150L single variant was most proficient at o-dianisidine oxidation with nitrite as the electron acceptor (8.5X wt). The library generation and screening method can be employed for evolving new reductase functions in NiR and for screening of efficient folding of engineered NiRs. Directed evolution of copper nitrite reductase to a chromogenic reductant.,MacPherson IS, Rosell FI, Scofield M, Mauk AG, Murphy ME Protein Eng Des Sel. 2010 Mar;23(3):137-45. Epub 2010 Jan 18. PMID:20083495 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Nitrite reductase 3D structures

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