Identification of a twin‐arginine leader‐binding protein

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (␣␤) 2 heterodimeric structure that is highly identical to homodimeric eukaryotic xanthine oxidoreductases. The crystal structure revealed that the molybdenum cofactor (Moco) is deeply buried within the protein. A protein involved in Moco insertion and XDH maturation has been identified, which was designated XdhC. XdhC was shown to be essential for the production of active XDH but is not a subunit of the purified enzyme. Here we describe the purification of XdhC and the detailed characterization of its role for XDH maturation. We could show that XdhC binds Moco in stoichiometric amounts, which subsequently can be inserted into Moco-free apo-XDH. A specific interaction between XdhC and XdhB was identified. We show that XdhC is required for the stabilization of the sulfurated form of Moco present in enzymes of the xanthine oxidase family. Our findings imply that enzymespecific proteins exist for the biogenesis of molybdoenzymes, coordinating Moco binding and insertion into their respective target proteins. So far, the requirement of such proteins for molybdoenzyme maturation has been described only for prokaryotes.Xanthine oxidoreductase is a complex metalloflavoprotein that catalyzes the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, using a water molecule as the ultimate source of oxygen incorporated into the product (1). The enzyme exists in two forms; the xanthine dehydrogenase form (XDH; EC1.17.1.4) 2 uses NAD ϩ as electron acceptor, whereas the xanthine oxidase (EC1.17.3.2) form uses O 2 as electron acceptor (2). Xanthine oxidoreductases are found both in eukaryotes and prokaryotes, with the enzymes isolated from bovine milk and Rhodobacter capsulatus being functionally and structurally the best characterized (3, 4). R. capsulatus XDH is a cytoplasmic enzyme with an (␣␤) 2 heterodimeric structure, with the two subunits encoded by the xdhA and xdhB genes, respectively (5 This report describes the detailed analysis for the requirement of XdhC to produce active XDH during heterologous expression in E. coli TP1000 cells. Analysis of XDH expressed under different aeration levels in the presence or absence of XdhC showed that, especially under aerobic conditions, XdhC is required to produce active XDH containing the terminal sulfur ligand of Moco. In addition, we purified and characterized XdhC after heterologous expression in E. coli. We could show that XdhC binds Moco/MPT in stoichiometric amounts and is able to insert bound Moco into Moco-free apo-XDH. In addition, a specific interaction between XdhC and XdhB was identified. We showed that XdhC acts as a Moco-binding protein, which protects the sulfurated form of Moco from oxidation. We propose that sulfurated Moco is inserted into apo-XDH by the aid of XdhC to produce active XDH. This is the first example of a system-specific protein involved in maturation of a molybdoenzyme for which Moco binding could be shown. Bacterial Strains, Pla...

Section: Discussionmentioning

confidence: 99%

Rhodobacter capsulatus XdhC Is Involved in Molybdenum Cofactor Binding and Insertion into Xanthine Dehydrogenase

Neumann¹,

Schulte²,

Jünemann³

et al. 2006

“…In the membrane, it associates with DmsC to form a functional membrane integral DmsABC trimer (26,27). DmsA signal sequences are bound by a specific chaperone, DmsD, which has been the first specific Tat substrate chaperone reported (5). We analyzed the presence of DmsA in membranes from the above described single and double mutant strains (Fig.…”

Section: Dnak and Slyd Have No Effects Or Have Only Minor Effects On mentioning

confidence: 99%

DnaK Plays a Pivotal Role in Tat Targeting of CueO and Functions beside SlyD as a General Tat Signal Binding Chaperone

Graubner

Schierhorn

Brüser

2007

“…The Tat translocase of Escherichia coli appears to be represented by ϳ600-kDa complexes that, depending on the detergent used for solubilization, contain varying amounts of TatA, TatB, and TatC (11)(12)(13). In addition, one RR signal sequence-binding protein, unrelated to TatABC, has been described (14). By use of a cell-free system with unprecedented high efficiency, we show here that in E. coli, translocation of Tat substrates across the cytoplasmic membrane follows a targeting step that is independent of the H ϩ gradient but requires an intact RR signal.…”

mentioning

confidence: 99%

Separate Analysis of Twin-arginine Translocation (Tat)-specific Membrane Binding and Translocation in Escherichia coli

Alami¹,

Trescher²,

Wu³

et al. 2002

The twin-arginine translocation (Tat) pathway exports those precursor proteins to the periplasmic space of bacteria that harbor a twin-arginine (RR) consensus motif in their signal sequences. We have reproduced translocation of several Tat substrates into inside-out plasma membrane vesicles from Escherichia coli. Translocation proceeding at an efficiency of up to 20% occurs specifically via the Tat pathway as indicated by (i) its requirement for elevated levels of the TatABC proteins in the membrane vesicles, (ii) competition by an intact twin-arginine signal peptide, and (iii) susceptibility toward dissipation of the transmembrane H ؉ gradient. The latter treatment, while blocking translocation, still allows for functional membrane association of Tat precursors. This is shown by the finding that translocation of isolated membrane-bound Tat precursor is restored upon re-energization of the vesicles.