Mutation of the conserved Cys165 outside of the CuA domain destabilizes nitrous oxide reductase but maintains its catalytic activity. Evidence for disulfide bridges and a putative protein disulfide isomerase gene.
The single conserved Cys165 outside of the CuA domain of nitrous oxide reductase (N2OR) from Pseudomonas stutzeri was mutated to glycine to test its presumed function in metal coordination of the catalytic site, CuZ. The point mutation reduced the cellular level of N2OR 5--10-fold compared to the level of the control strain. In the mutant, the activity and the Cu content of the enzyme, as well as the transcript level of the N2OR structural gene, nosZ, remained unaffected. The mutant enzyme was processed and exported into the periplasm like the wild-type enzyme. Chemical analysis for sulfhydryl groups gave about nine -SH groups/monomer of the apoenzyme prepared from the wild-type enzyme, in accordance with the nine cysteine residues of the derived amino acid sequence. Eight -SH groups were found to form disulfide bridges in the holoenzyme dimer. We propose that in the native state of the enzyme Cys165 does not bind to CuZ, but may be part of a disulfide bridge essential for the stability of N2OR. Immediately downstream of the genes nosDFY, encoding the components for Cu incorporation into the reductase, we have identified the open reading frame, ORFL, whose derived product has the signature of a protein disulfide isomerase.
Dreusch
A
A
Lehrstuhl für Mikrobiologie, Universität Fridericiana, Karlsruhe, Germany.
Riester
J
J
Kroneck
P M
PM
Zumft
W G
WG
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D005798:Genes, Bacterial; D007535:Isomerases / Q000235:genetics*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D019704:Protein Disulfide-Isomerases; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004269:DNA, Bacterial; D004220:Disulfides; D010088:Oxidoreductases; C022763:nitrous oxide reductase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D003545:Cysteine