Transfer of the Molybdenum Cofactor Synthesized by Rhodobacter capsulatus MoeA to XdhC and MobA

Abstract

The molybdenum cofactor (Moco) 2 is an essential component of a diverse group of enzymes involved in important redox reactions in the global carbon, nitrogen, and sulfur cycles. Moco consists of a molybdenum atom coordinated to the dithiolene group of a tricyclic pyranopterin referred to as molybdopterin (MPT) (1). The biosynthesis of Moco is highly conserved in eukaryotes and prokaryotes (1) and can be divided into three general steps. In the first step, GTP is converted to the meta-stable intermediate Precursor Z (2, 3). In the second step, Precursor Z is further transformed by MPT synthase into MPT by generation of its characteristic dithiolene group (4, 5). In the third step, molybdate is inserted to the MPT dithiolene sulfurs, a reaction catalyzed by MogA and MoeA in Escherichia coli (6 -8). MoeA mediates molybdenum ligation, whereas MogA helps to facilitate this step in an ATP-dependent manner (8). Recently, studies with the homologous Arabidopsis thaliana CNX1 protein G and E domains identified the formation of an MPT-AMP intermediate before the ligation of molybdate to the MPT moiety (9, 10). An unexpected observation in the crystal structure of the A. thaliana CNX1 protein G domain was the identification of copper bound to the MPT-AMP dithiolene sulfurs (11). Up to now, the function of this novel MPT ligand has been unknown, but it was speculated that copper might play a role in sulfur transfer to Precursor Z, in protection of the MPT dithiolene from oxidation, and/or in presentation of a suitable leaving group for molybdenum insertion (12). To date, copper-MPT-AMP has not been identified as an intermediate in the biosynthesis of Moco in E. coli (8).After the insertion of molybdenum into MPT in E. coli, Moco either can be directly inserted into molybdoenzymes (such as YedY) binding the MPT form of Moco (13) or is further modified by attachment of GMP (14, 15), forming the bis-MPT guanine dinucleotide (bis-MGD) form of Moco found in enzymes of the Me 2 SO reductase family (16). In E. coli, the GMP attachment to Moco is catalyzed by the MobA and MobB proteins (17). Whereas MobA was shown to be essential for this reaction (18), the role of MobB still remains uncertain. From the crystal structure, it was postulated that MobB is an adapter protein that acts in concert with MobA to achieve the efficient biosynthesis and utilization of MGD (19).Enzymes containing the MPT form of Moco belong to either the sulfite oxidase or xanthine oxidase family, whereas enzymes binding the bis-MGD form of Moco belong to the Me 2 SO reductase family of molybdoenzymes. In Rhodobacter capsulatus, xanthine dehydrogenase (XDH; EC 1.17.1.4) is the only identified enzyme harboring the MPT form of the cofactor, whereas all other known molybdoenzymes bind the bis-MGD form of the cofactor (20). An essential role for the XdhC protein in the maturation of R. capsulatus XDH has been described, which entails binding of Moco and its insertion into the XdhB subunit of XDH (21). For all members of the xanthine oxidase family, the sulfurate...

The Mechanism of Assembly and Cofactor Insertion into Rhodobacter capsulatus Xanthine Dehydrogenase

“…7). The biosynthesis of Moco is additionally a complex process involving more than a dozen different proteins (27), with the insertion of sulfurated Moco by the XdhC protein being the last step of XDH maturation (28). This step is strictly regulated in R. capsulatus, because in vivo di-oxo Moco is not inserted into R. capsulatus XDH (29).…”

Specific Interactions between Four Molybdenum-Binding Proteins Contribute to Mo-Dependent Gene Regulation in Rhodobacter capsulatus

“…As a reference, the multielement standard XVI (Merck, Darmstadt, Germany) was used. Bovine serum albumin and R. capsulatus MoeA and MogA (22) were used for control experiments. (Fig.…”

Dedicated Metallochaperone Connects Apoenzyme and Molybdenum Cofactor Biosynthesis Components

“…To construct plasmid pMobA, which allows the synthesis of His 6 -tagged MobA, the same cloning strategy as that described previously (14) was used except that the mobA coding sequence was cloned into the BamHI-HindIII cloning sites of pet21(ϩ) (Novagen). Plasmid pMN56 allowing R. capsulatus MobA production was described previously (28). Transformations were carried out according to the method of Chung and Miller (29).…”

“…The phosphate monoester of Form A was cleaved by the addition of 40 mM MgCl 2 and 1 unit of calf intestine alkaline phosphatase. MGD was extracted from TorD and converted to Form A-GMP by overnight treatment with acidic iodine at room temperature as described previously (28). Form A-GMP was purified by chromatography on Q-Sepharose (GE Healthcare).…”

“…Binding of MGD-MobA was expressed from plasmid pMN56 and purified as described earlier (28). The His tag was removed by usage of the Thrombin CleanCleave kit (Sigma).…”