Lack of copper insertion into unprocessed cytoplasmic nitrous oxide reductase generated by an R20D substitution in the arginine consensus motif of the signal peptide

Dreusch, Andreas; Bürgisser, Daniel; Heizmann, Claus W.; Zumft, Walter G.

doi:10.1016/s0005-2728(96)00174-0

Cited by 49 publications

(45 citation statements)

References 10 publications

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“…A similar result was achieved by reconstitution of purified nitrous oxide reductase from which the copper centers had been chemically removed (8). In contrast, the characteristic electronic spectrum of Cu A could be obtained when a translocationincompetent mutant of nitrous oxide reductase in which the twin-arginine motif was mutated (R20D) was reconstituted with copper, but the insertion process was slow and resulted in a weak retention of copper (11). These data led to the suggestion that the twin-arginine motif of NosZ may confer both translocational competence and delivery to a specific system involved with copper insertion.…”

Section: Discussionmentioning

confidence: 73%

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

et al. 2000

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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.

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Section: Discussionmentioning

confidence: 73%

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

et al. 2000

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“…that the mislocalization of NosZ in TF140 impairs its physiological function. Indeed, mutational analysis of the NosZ system from Pseudomonas stutzeri has shown that the export to the periplasm is a prerequisite of the protein to be physiologically active (11). Due to the lack of an appropriate NosZ antibody, it was not possible to investigate the cellular localization of the enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…The primary structures of NosZ from various bacteria, including R. eutropha, are highly conserved and are all characterized by an unusually long N-terminal twin-arginine signal peptide of 45 to 56 aa (11,47).…”

mentioning

confidence: 99%

Ralstonia eutropha TF93 Is Blocked in Tat-Mediated Protein Export

2000

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Ralstonia eutropha (formerly Alcaligenes eutrophus) TF93 is pleiotropically affected in the translocation of redox enzymes synthesized with an N-terminal signal peptide bearing a twin arginine (S/T-R-R-X-F-L-K) motif. Immunoblot analyses showed that the catalytic subunits of the membrane-bound [NiFe] hydrogenase (MBH) and the molybdenum cofactor-binding periplasmic nitrate reductase (Nap) are mislocalized to the cytoplasm and to the inner membrane, respectively. Moreover, physiological studies showed that the coppercontaining nitrous oxide reductase (NosZ) was also not translocated to the periplasm in strain TF93. The cellular localization of enzymes exported by the general secretion system was unaffected. The translocationarrested MBH and Nap proteins were enzymatically active, suggesting that twin-arginine signal peptidedependent redox enzymes may have their cofactors inserted prior to transmembrane export. The periplasmic destination of MBH, Nap, and NosZ was restored by heterologous expression of Azotobacter chroococcum tatA mobilized into TF93. tatA encodes a bacterial Hcf106-like protein, a component of a novel protein transport system that has been characterized in thylakoids and shown to translocate folded proteins across the membrane.Periplasmic enzymes binding redox cofactors play a central role in alternative energy metabolism of gram-negative bacteria. In contrast to cytochrome c-type proteins, various periplasmic enzymes binding constituents, such as the molybdenum cofactor, a [NiFe] site, copper centers, or iron-sulfur clusters, contain a conserved, positively charged -S/T-RRXFLK-(twinarginine) element within their N-terminal signal peptides, pointing to a special translocation pathway (2). Very recently, a system responsible for the membrane targeting and translocation of [NiFe] hydrogenases, as well as the molybdenum enzymes dimethylsulfoxide reductase, trimethylamine N-oxide reductase, and periplasmic nitrate reductase, has been characterized for Escherichia coli (32,43). Mutant analyses suggested that the translocation of the twin-arginine signal peptide-bearing enzymes proceeds independently of the general secretion machinery of the cell (Sec), presumably via intermediates which have their cofactors inserted (31).Ralstonia eutropha (formerly Alcaligenes eutrophus [7]) is the host of at least three periplasmic cofactor-containing enzymes synthesized with an N-terminal twin-arginine signal peptide: the membrane-bound hydrogenase (MBH) (involved in energy generation from H 2 ), the periplasmic nitrate reductase (Nap) (reduces nitrate to nitrite), and the nitrous oxide reductase (NosZ) (a component of the denitrification pathway). All three enzymes are encoded in R. eutropha H16 by megaplasmidborne genes.The MBH of R. eutropha is a member of the [NiFe] hydrogenases (15) composed of a heterodimer (HoxKG) attached to the periplasmic surface of the inner membrane by a cytochrome b-type anchor protein (HoxZ) (4, 13). Hydrogen is activated at the [NiFe] site of HoxG (62 kDa), and the electrons are tra...

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“…Some examples of Tat targeting signals are shown in Fig. 2. A number of site-directed mutagenesis experiments using natural substrates of the Tat pathway have confirmed that the consecutive arginine residues of the consensus motif play a key role in the export process (Dreusch et al, 1997;Gross et al, 1999;Halbig et al, 1999). However, there is growing evidence that Tat translocation does not always require the presence of an arginine pair.…”

Section: The Tat Signal Peptidementioning

confidence: 99%

Moving folded proteins across the bacterial cell membrane

Palmer

Berks

2003

Microbiology

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The Tat protein export system is located in the bacterial cytoplasmic membrane and operates in parallel to the well-known Sec pathway. While the Sec system only transports unstructured substrates, the function of the Tat pathway is to translocate folded proteins. The Tat translocase thus faces the formidable challenge of moving structured macromolecular substrates across the bacterial cytoplasmic membrane without rendering the membrane freely permeable to protons and other ions. The substrates of the Tat pathway are often proteins that bind cofactor molecules in the cytoplasm, and are thus folded, prior to export. Such periplasmic cofactor-containing proteins are essential for most types of bacterial respiratory and photosynthetic energy metabolism. In addition, the Tat pathway is involved in outer membrane biosynthesis and in bacterial pathogenesis. Substrates are targeted to the Tat pathway by amino-terminal signal sequences harbouring consecutive, essentially invariant, arginine residues, and movement of proteins through the Tat system is energized by the transmembrane proton electrochemical gradient. The TatA protein probably forms the transport channel while the TatBC proteins act as a receptor complex that recognizes the signal peptide of the substrate protein.

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Lack of copper insertion into unprocessed cytoplasmic nitrous oxide reductase generated by an R20D substitution in the arginine consensus motif of the signal peptide

Cited by 49 publications

References 10 publications

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

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

Ralstonia eutropha TF93 Is Blocked in Tat-Mediated Protein Export

Moving folded proteins across the bacterial cell membrane

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