Eur. J. Biochem. zyxwvutsrqpo 2I8, 49-57 (1993) zyxwvuts 0 FEBS 1993 zyxwvutsrqp Sequence and expression of the gene encoding the respiratory nitrous-oxide reductase from zyxw Paracoccus denitrificans New and conserved structural and regulatory motifs Frank U. HOEREN', Ben C. BERKS*, Stuart J. FERGUSON' and John E. G. McCARTHY' Department of Gene Expression, Gesellschaft fur Biotechnologische Forschung, Braunschweig, Germany Department of Biochemistry, University of Oxford, England (Received June 29/August 10,1993) zyxwvutsr - EJB 93 0970/2 The structural gene for the respiratory nitrous-oxide reductase from Paracoccus denitrijicans has been cloned using a probe derived from the structural gene, nosZ, for this enzyme from Pseu- domonas stutzeri. The cloned gene could be expressed surprisingly well (presumably yielding an apo-protein) using an expression vector in Escherichia coli. Sequencing the nos2 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. denitrifcans 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: denitrifcans 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 trypto- phan 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 Cu, region of subunit zyxwv I1 of the cytochrome aa3 from P. denit@- cans 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 l? 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 transcriptiodtranslation system was developed for l? denitrificans which permit- ted the expression of the cloned gene for nitrous-oxide reductase and which will be of general value in other studies of this organism. In bacterial denitrification, respiratory electron transport terminates with the reduction of nitrate, nitrite, nitric oxide and nitrous-oxide (Ferguson, 1987; Zumft, 1992). Thus the overall process leads to the conversion of nitrate to nitrogen gas with the concomitant synthesis of ATP (Nicholls and Fer- Correspondence to J. E. G. McCarthy, Department of Gene Ex- pression, Gesellschaft fur Biotechnologische Forschung, Masche- roder Weg 1, D-38124Braunschweig, Germany Fax: +49 531 6181 458. Abbreviation. FNR box, binding site for a trans-acting regulatory protein (Fnr) that activates gene expression under anaerobic condi- tions; ANR box, the equivalent to FNR box in P. aeruginosa. Enzyme. Nitrous-oxide reductase (EC 1.7.99.6). Note. The novel nucleotide sequence data published here have been submitted to the EMBL sequence data bank(s) and are avail- able under accession number(s) X74792 PONORNOZ. guson, 1992). The enzyme catalysing the final step in this sequence, nitrous-oxide reductase, was first purified from Pseudomonas stutzeri (Zumft and Matsubara, 1982; Coyle et al., 1985). Subsequently, the same type of periplasmic en- zyme (Boogerd et al., 1981; Alefounder et al., 1983; Vie- brock and Zumft, 1988) has been shown to occur in a number of other organisms, including Paracoccus denitrificans (Sny- der and Hollocher, 1987) and the non-denitrifying bacterium Rhodobacter capsulatus (McEwan et al., 1985). This nitrous- oxide reductase is a dimeric protein in which each polypep- tide chain most probably contains four atoms of copper. A variety of spectroscopic techniques has shown that there ex- ists within the enzyme a copper centre that is very similar to the Cu, centre found in cytochrome aa3 oxidase. Although once considered to contain just one copper, this centre is now thought to be a binuclear copper site (Antholine et al., 1992).