NDLI: The Blue Copper-Containing Nitrite Reductase from Alcaligenes xylosoxidans: Cloning of the nirA Gene and Characterization of the Recombinant Enzyme

Content Provider	PubMed Central
Author	Miguel, Prudêncio Eady, Robert R. Sawers, Gary
Copyright Year	1999
Abstract	The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed in Escherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.
Starting Page	2323
File Format	PDF
ISSN	10985530
e-ISSN	10985530
Journal	Journal of Bacteriology
Issue Number	8
Volume Number	181
Language	English
Publisher	American Society for Microbiology
Publisher Date	1999-04-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Microbiology

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Purification and characterization of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015): evidence for the presence of both type 1 and type 2 copper centres

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