Lack of copper insertion into unprocessed cytoplasmic nitrous oxide reductase generated by an R20D substitution in the arginine consensus motif of the signal peptide.
Metal insertion into an engineered cytoplasmic form of the multicopper enzyme N2O reductase (N2OR) (EC 1.7.99.6) of Pseudomonas stutzeri was studied. The reductase has an unusually long presequence of 50 amino acids for translocation into the periplasm. The signal peptide of N2OR shares a conserved twin-arginine sequence motif with the signal peptides of other N2O reductases and a sizeable group of periplasmic or membrane-bound enzymes, requiring cofactor insertion or processing. A catalytically inactive reductase, N2ORR20D, that lacked Cu, accumulated in the cytoplasm on mutation of the first arginine of this motif. The CuA site of N2ORR20D could be reconstituted in vitro indicating that the lack of metal was not due to a serious conformational restraint. Our findings locate the event of in vivo Cu insertion into N2OR in the periplasm or allow it to take place concomitant with protein translocation.
Dreusch
A
A
Lehrstuhl für Mikrobiologie, Universität Fridericiana, Karlsruhe, Germany.
Bürgisser
D M
DM
Heizmann
C W
CW
Zumft
W G
WG
D000595:Amino Acid Sequence; D001120:Arginine; D001224:Aspartic Acid; D016384:Consensus Sequence; D003300:Copper / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D017931:DNA Primers; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D001224:Aspartic Acid; D003300:Copper; D001120:Arginine; D010088:Oxidoreductases; C022763:nitrous oxide reductase