Sequence and expression of the gene encoding the respiratory nitrous‐oxide reductase from Paracoccus denitrificans

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Abstract
The structural gene for the respiratory nitrous-oxide reductase from Paracoccus denitrificans has been cloned using a probe derived from the structural gene, nosZ, for this enzyme from Pseudomonas stutzeri. The cloned gene could be expressed surprisingly well (presumably yielding an apo-protein) using an expression vector in Escherichia coli. Sequencing the nosZ gene from P. denitrificans has shown that the periplasmic nitrous-oxide reductase of this organism is highly similar in sequence to previously derived primary sequences for the enzyme from three other organisms. As with the other reductases, an unusually long signal sequence is deduced and a common motif of GXXRRXXLG near the beginning of this sequence is present. The results of N-terminal sequencing of the mature nitrous-oxide reductase from the closely related organism Thiosphaera pantotropha indicate that processing of the P. denitrificans precursor occurs between amino acids at positions 57 and 58. The predicted signal peptide is therefore of the same length and of similar overall structure to that previously described for the P. denitrificans methylamine dehydrogenase small subunit (MauA). The P. denitrificans sequence for the mature nitrous-oxide reductase reduces from 14 to 11 and 6 to 4, respectively, the number of conserved histidine and methionine residues compared to previous sequences. Three cysteine and four tryptophan residues, previously identified as conserved amongst nitrous-oxide reductases, are found in the Paracoccus enzyme. A comparison of the sequence of the C-terminal region of the nitrous-oxide-reductase sequence with that for the CuA region of subunit II of the cytochrome aa3 from P. denitrificans reveals considerable sequence similarities. Upstream of the structural gene for nosZ are sequences TTGAAGCTTAACCAG (centred at position -21 with respect to the start codon) and CCCGGTGGTCATCAAG (centred at position -126). Although both could be FNR (ANR) boxes, the latter is far more probable to have this role because only it is likely to be upstream of a promoter site. This is the first indication at the DNA sequence level for the existence of this regulatory system in P. denitrificans. Analysis of the flanking DNA sequences revealed reading frames upstream and downstream of the nosZ gene showing similarity to the nosR and nosD genes, respectively, of Pseudomonas species. An S30 in vitro transcription/translation system was developed for P. denitrificans which permitted the expression of the cloned gene for nitrous-oxide reductase and which will be of general value in other studies of this organism.