3H4F

Met62Leu variant of nitrite reductase from Alcaligenes faeclis

3H4F の概要

• エントリーDOI: 10.2210/pdb3h4f/pdb
• 関連するPDBエントリー: 1SJM 3H4H
• 分子名称: Copper-containing nitrite reductase, COPPER (II) ION (3 entities in total)
• 機能のキーワード: nitrite reductase, nir, high-throughput screening, oxidase, copper, fad, flavoprotein, metal-binding, nitrate assimilation, oxidoreductase, periplasm, pyrrolidone carboxylic acid
• 由来する生物種: Alcaligenes faecalis
• 細胞内の位置: Periplasm: P38501
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 109290.25

構造登録者

MacPherson, I.S.,Rosell, F.I.,Scofield, M.,Mauk, A.G.,Murphy, M.E.P. (登録日: 2009-04-19, 公開日: 2010-02-02, 最終更新日: 2023-09-06)

主引用文献

Macpherson, I.S.,Rosell, F.I.,Scofield, M.,Mauk, A.G.,Murphy, M.E.
Directed evolution of copper nitrite reductase to a chromogenic reductant.
Protein Eng.Des.Sel., 23:137-145, 2010

PubMed Abstract: 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.

PubMed: 20083495
DOI: 10.1093/protein/gzp084

実験手法

X-RAY DIFFRACTION (2.1 Å)