Document Detail

Directed evolution of copper nitrite reductase to a chromogenic reductant.
MedLine Citation:
PMID:  20083495     Owner:  NLM     Status:  MEDLINE    
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.
Iain S MacPherson; Federico I Rosell; Melanie Scofield; A Grant Mauk; Michael E P Murphy
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2010-01-18
Journal Detail:
Title:  Protein engineering, design & selection : PEDS     Volume:  23     ISSN:  1741-0134     ISO Abbreviation:  Protein Eng. Des. Sel.     Publication Date:  2010 Mar 
Date Detail:
Created Date:  2010-02-05     Completed Date:  2010-06-28     Revised Date:  2013-05-31    
Medline Journal Info:
Nlm Unique ID:  101186484     Medline TA:  Protein Eng Des Sel     Country:  England    
Other Details:
Languages:  eng     Pagination:  137-45     Citation Subset:  IM    
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
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MeSH Terms
Alcaligenes faecalis / enzymology,  genetics
Azurin / metabolism
Chromogenic Compounds / metabolism*
Copper / metabolism
Crystallography, X-Ray
Dianisidine / metabolism
Directed Molecular Evolution*
Enzyme Assays
High-Throughput Screening Assays
Models, Molecular
Mutagenesis, Site-Directed
Nitrite Reductases / chemistry,  genetics*,  isolation & purification,  metabolism*
Oxygen / metabolism
Protein Conformation
Reducing Agents / metabolism*
Reproducibility of Results
Spectrum Analysis
Reg. No./Substance:
0/Chromogenic Compounds; 0/Reducing Agents; 0/pseudoazurin; 119-90-4/Dianisidine; 12284-43-4/Azurin; 7440-50-8/Copper; 7782-44-7/Oxygen; EC 1.7.-/Nitrite Reductases; EC reductase, copper-containing

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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