The NosX and NirX Proteins of Paracoccus denitrificans Are Functional Homologues: Their Role in Maturation of Nitrous Oxide Reductase

Abstract

The nos (nitrous oxide reductase) operon of Paracoccus denitrificans contains a nosX gene homologous to those found in the nos operons of other denitrifiers. NosX is also homologous to NirX, which is so far unique to P. denitrificans. Single mutations of these genes did not result in any apparent phenotype, but a double nosX nirX mutant was unable to reduce nitrous oxide. Promoter-lacZ assays and immunoblotting against nitrous oxide reductase showed that the defect was not due to failure of expression of nosZ, the structural gene for nitrous oxide reductase. Electron paramagnetic resonance spectroscopy showed that nitrous oxide reductase in cells of the double mutant lacked the Cu A center. A twin-arginine motif in both NosX and NirX suggests that the NosX proteins are exported to the periplasm via the TAT translocon.

Alternative Pyrimidine Biosynthesis Protein ApbE Is a Flavin Transferase Catalyzing Covalent Attachment of FMN to a Threonine Residue in Bacterial Flavoproteins

“…Simultaneous disruption of the genes encoding two ApbE orthologs (NosX and NirX; Fig. 2) in P. denitrificans cells eliminates their NO reductase activity (47), which can now be explained by defective maturation of the regulator of NO reductase transcription NosR, which contains a covalently bound FMN residue (16).…”

Requirements for Cu A and Cu-S Center Assembly of Nitrous Oxide Reductase Deduced from Complete Periplasmic Enzyme Maturation in the Nondenitrifier Pseudomonas putida

“…NosX proteins exhibit high sequence similarity among themselves but not with other proteins (46), although sequence similarity to RnfF has been noted (6). RnfF is a membranebound periplasmic protein belonging to an R. capsulatus complex presumably involved in electron transport for nitrogen fixation (47).…”

“…The precise role of this gene in NosZ biosynthesis has not been clarified, although it is necessary for N 2 O utilization by Sinorhizobium meliloti (6) and Paracoccus denitrificans (46). The latter bacterium carries in addition to nosX a homologous gene, nirX, and mutagenesis of both genes is required to generate a Nos Ϫ phenotype.…”

Lack of the ApbC or ApbE Protein Results in a Defect in Fe-S Cluster Metabolism in Salmonella enterica Serovar Typhimurium

“…ApbE was subsequently shown to be a periplasmic lipoprotein whose location but not membrane association was required for efficient thiamine synthesis (1,2). Mutations in genes similar to apbE have been isolated in various organisms and result in a lack of nitrous oxide reductase activity (nosX mutants) in Paracoccus denitrificans (47) and Rhizobium meliloti (11) and a defect in nitrogen fixation (rnfF mutants) in Rhodobacter capsulatus (48).…”