Abstract
The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of “Achromobacter cycloclastes” nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd 1 nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d 1, indicating that both heterologous hosts synthesized a cytochrome cd 1 without the d 1-group.
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Abbreviations
- Cu-NIR:
-
Cu-containing nitrite reductase
- DDC:
-
diethyldithiocarbamate
- EPR:
-
electron paramagnetic resonance
- IPTG:
-
isopropyl-β-D-galactoside
- SDS:
-
sodium dodecyl sulfate
- LB medium:
-
Luria-Bertani medium
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Glockner, A.B., Jüngst, A. & Zumft, W.G. Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd 1-free background (NirS−) of Pseudomonas stutzeri . Arch. Microbiol. 160, 18–26 (1993). https://doi.org/10.1007/BF00258141
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DOI: https://doi.org/10.1007/BF00258141