3h4f

Met62Leu variant of nitrite reductase from Alcaligenes faeclis

spinqualitylabelsall models 3h4f, resolution 2.10Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3h4f is a 3 chain structure with sequence from Alcfa. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: CU Related: 1sjm, 3h4h Gene: nir, nirK (ALCFA) Activity: Nitrite reductase (NO-forming), with EC number 1.7.2.1 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT 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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