ArticleProtein engineering, design & selection : PEDS2010
Directed evolution of copper nitrite reductase to a chromogenic reductant.
Article in Protein engineering, design & selection : PEDS, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed, 24 citations in OpenAlex.
- Article
- The heme-sensitive regulator SbnI has a bifunctional role in staphyloferrin B production byThe Journal of biological chemistry · 2019Article
- Engineering a bifunctional copper site in the cupredoxin fold by loop-directed mutagenesis.Chemical science · 2018Article
- SbnI is a free serine kinase that generatesThe Journal of biological chemistry · 2018Article
- Structure-function analyses reveal key features inThe Journal of biological chemistry · 2018Article
- Article
- New molecular packing in a crystal of pseudoazurin from Alcaligenes faecalis: a double-helical arrangement of blue copper.Acta crystallographica. Section F, Structural biology communications · 2017Article
- Redox-coupled structural changes in nitrite reductase revealed by serial femtosecond and microfocus crystallography.Journal of biochemistry · 2016Article
- A Diatom Ferritin Optimized for Iron Oxidation but Not Iron Storage.The Journal of biological chemistry · 2015Article
- SbnG, a citrate synthase in Staphylococcus aureus: a new fold on an old enzyme.The Journal of biological chemistry · 2014Article
- IruO is a reductase for heme degradation by IsdI and IsdG proteins in Staphylococcus aureus.The Journal of biological chemistry · 2013Article
- Characterization of a nitrite reductase involved in nitrifier denitrification.The Journal of biological chemistry · 2013Article
- Inactivation of the heme degrading enzyme IsdI by an active site substitution that diminishes heme ruffling.The Journal of biological chemistry · 2012Article
- Biocatalyst development by directed evolution.Bioresource technology · 2012Review
- Unique heme-iron coordination by the hemoglobin receptor IsdB of Staphylococcus aureus.Biochemistry · 2011Article
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Authors and funding
5 authors at 1 institution in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
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.
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