The synthesis of a functional nitrous oxide reductase requires an assembly apparatus for the insertion of the prosthetic copper. Part of the system is encoded by maturation genes located in Pseudomonas stutzeri immediately downstream of the structural gene for the enzyme. We have studied the transcriptional organization and regulation of this region and found a nosDFYL tatE operon structure. In addition to a putative ABC transporter, consisting of NosD, NosF, and NosY, the operon encodes a Cu chaperone, NosL, and a component of the Tat translocon, TatE. The nosD operon was activated in response to anaerobiosis and nitrate denitrification. The membrane-bound regulator NosR was required for operon expression; in addition, DnrD, a regulator of the Crp-Fnr family, enhanced expression under anaerobic conditions. This establishes a likely signal transduction sequence of NO 3 DnrD 3 nosR/NosR 3 nosD operon. DnrD-dependent expression was also observed for the nnrS operon (located immediately downstream of the nosD operon), which encodes a putative heme-Cu protein (NnrS) and a member of the short-chain dehydrogenase family (ORF247). The NosF protein, encoded within the nosD operon, exhibits sequence similarity to ABC-type ATPases. It was fused to the Escherichia coli maltose-binding protein and overexpressed in soluble form. The fusion protein was purified and shown to have ATPase activity. NosF is the first maturation factor for which a catalytic function has been demonstrated in vitro.The multicopper enzyme nitrous oxide reductase (N 2 OR) undergoes a maturation process for the insertion of its prosthetic metal (32,39,44,48,49). Several accessory proteins are required for the biosynthesis of the catalytically active enzyme, some of which are encoded by genes located downstream of the N 2 OR structural gene, nosZ. Expression of nosZ is dependent on the multitopic, membrane-bound regulator NosR (10). At least three proteins, encoded by the maturation genes nosD, -F, and -Y, form a putative assembly complex which extends to both sides of the cytoplasmic membrane. From sequence similarity, it was inferred that nosF may encode an ATP/GTPase and that a step in the biosynthesis of N 2 OR is energy dependent (49). A lacZ reporter gene fusion had been used to deduce a location in the cytoplasm for NosF (9). NosD belongs to a family of proteins with a carbohydrate binding and sugar hydrolase domain (8) whose significance is still unclear. NosY is a six-helix transmembrane protein; it is presumed that, together with NosF and periplasmic NosD, it forms an ABC-type transporter. The biosynthesis of N 2 OR also involves a number of additional factors which are nonessential or can be functionally replaced by other cellular processes (44).Catalyzed assembly of the prosthetic Cu in N 2 OR seems to be obligatory for the Cu Z center, representing the catalytic site in the form of a tetranuclear CuS cluster (6, 35), rather than for the binuclear Cu A center, which represents the electron entry site (27). Mutational inactivation of any of t...
By transforming N 2 O to N 2 , the multicopper enzyme nitrous oxide reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. The signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the Tat pathway. We have identified tat genes of Pseudomonas stutzeri and functionally analyzed the unlinked tatC and tatE loci. A tatC mutant retained N 2 O reductase in the cytoplasm in the unprocessed form and lacking the metal cofactors. This is contrary to viewing the Tat system as specific only for fully assembled proteins. A C618V exchange in the electron transfer center Cu A rendered the enzyme largely incompetent for transport. The location of the mutation in the C-terminal domain of N 2 O reductase implies that the Tat system acts on a completely synthesized protein and is sensitive to a late structural variation in folding. By generating a tatE mutant and a reductase-overproducing strain, we show a function for TatE in N 2 O reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite reductase system. The cytochrome cd 1 nitrite reductase was found in the periplasm of the tatC mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D 1 macrocycle. The NirD protein as part of a complex required for heme D 1 synthesis or processing carries a putative Tat signal peptide. Since NO reduction was also inhibited in the tatC mutant, the Tat protein translocation system is necessary in multiple ways for establishing anaerobic nitrite denitrification.
Erster Schritt bei der Strukturaufklärung von Proteinen mit Röntgenstrukturanalyse ist die Züchtung von Proteinkristallen. Hochdurchsatz‐Techniken sowie spezialisierte Mikroplatten tragen entscheidend dazu bei, sowohl den zeitlichen als auch den finanziellen Aufwand zu reduzieren.
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