In Vitro Incorporation of Nascent Molybdenum Cofactor into Human Sulfite Oxidase

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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: Analysis Of the Ability Of Xdhc To Insert Moco Intomentioning

confidence: 72%

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

Neumann¹,

Schulte²,

Jünemann³

et al. 2006

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“…The MPT-Mo cofactor can either be inserted into MPT-free apoenzymes or undergo subsequent addition of a nucleotide, for example GMP in E. coli by the MobA protein. However, in vitro reconstitution studies of MPT-enzymes such as sulfite oxidase have indicated that conversion of MPT to active Moco by molybdate chelation and its subsequent incorporation can be performed in the absence of MogA and MoeA (13). Similar studies performed on MGDenzymes such as Me 2 SO reductase from Rhodobacter sphaeroides have shown that both MobB and a chaperone protein are not absolutely required for MGD insertion (15).…”

mentioning

confidence: 67%

“…The crystal structure of most of the proteins involved in the biosynthetic pathway of Moco have been determined recently (4 -12). Biochemical studies have indicated that newly formed MPT remains tightly bound to the MoaD-MoaE complex (MPT synthase) until its transfer to proteins able to bind it with higher affinity (13). MogA and MoeA proteins constitute such candidates and have been shown to bind MPT with distinct affinities (6,11,14).…”

mentioning

confidence: 99%

In VivoInteractions between Gene Products Involved in the Final Stages of Molybdenum Cofactor Biosynthesis inEscherichia coli

Magalon

Frixon

Pommier

et al. 2002

The final stages of bacterial molybdenum cofactor (Moco) biosynthesis correspond to molybdenum chelation and nucleotide attachment onto an unique and ubiquitous structure, the molybdopterin. Using a bacterial two-hybrid approach, here we report on the in vivo interactions between MogA, MoeA, MobA, and MobB implicated in several distinct although linked steps in Escherichia coli. Numerous interactions among these proteins have been identified. Somewhat surprisingly, MobB, a GTPase with a yet unclear function, interacts with MogA, MoeA, and MobA. Probing the effects of various mo. mutations on the interaction map allowed us (i) to distinguish Moco-sensitive interactants from insensitive ones involving MobB and (ii) to demonstrate that molybdopterin is a key molecule triggering or facilitating MogA-MoeA and MoeA-MobA interactions. These results suggest that, in vivo, molybdenum cofactor biosynthesis occurs on protein complexes rather than by the separate action of molybdenum cofactor biosynthetic proteins.

“…Since tight binding of MPT to MoaE had been observed previously (9,28) (MoaE-MoaD-SH) were passed through a desalting column equilibrated in water and concentrated prior to negative ion mass spectroscopy.…”

Section: Resultsmentioning

confidence: 99%

Mechanistic and Mutational Studies of Escherichia coli Molybdopterin Synthase Clarify the Final Step of Molybdopterin Biosynthesis

Wuebbens

Rajagopalan

2003

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111

Biosynthesis of the molybdenum cofactor, a chelate of molybdenum or tungsten with a novel pterin, occurs in virtually all organisms including humans. In the cofactor, the metal is complexed to the unique cis-dithiolene moiety located on the pyran ring of molybdopterin. Escherichia coli molybdopterin synthase, the protein responsible for adding the dithiolene to a desulfo precursor termed precursor Z, is a dimer of dimers containing the MoaD and MoaE proteins. The sulfur used for dithiolene formation is carried in the form of a thiocarboxylate at the MoaD C terminus. Using an intein expression system for preparation of thiocarboxylated MoaD, the mechanism of the molybdopterin synthase reaction was examined.