Abstract
Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a molybdo-flavoprotein that is highly homologous to the homodimeric mammalian xanthine oxidoreductase. However, the bacterial enzyme has an (alphabeta)(2) heterotetrameric structure, and the cofactors were identified to be located on two different polypeptides. We have analyzed the mechanism of cofactor insertion and subunit assembly of R. capsulatus XDH, using engineered subunits with appropriate substitutions in the interfaces. In an (alphabeta) heterodimeric XDH containing the XdhA and XdhB subunits, the molybdenum cofactor (Moco) was shown to be absent, indicating that dimerization of the (alphabeta) subunits has to precede Moco insertion. In an (alphabeta)(2) XDH heterotetramer variant, including only one active Moco-center, the active (alphabeta) site of the chimeric enzyme was shown to be fully active, revealing that the two subunits act independent without cooperativity. Amino acid substitutions at two cysteine residues coordinating FeSI of the two [2Fe-2S] clusters of the enzyme demonstrate that an incomplete assembly of FeSI impairs the formation of the XDH (alphabeta)(2) heterotetramer and, thus, insertion of Moco into the enzyme. The results reveal that the insertion of the different redox centers into R. capsulatus XDH takes place sequentially. Dimerization of two (alphabeta) dimers is necessary for insertion of sulfurated Moco into apo-XDH, the last step of XDH maturation.
Highlights
Mammalian Xanthine oxidoreductases (XORs) catalyze the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, and exist originally as the dehydrogenase form (XDH, EC 1.17.1.4) but can be converted to the oxidase form (XO, EC 1.1.3.22) either reversibly by oxidation of sulfhydryl residues of the protein molecule or irreversibly by proteolysis [4]
The amino acid sequence of R. capsulatus xanthine dehydrogenase (XDH) has a high degree of similarity to eukaryotic XORs, in contrast to the homodimeric (␣)2 structure of eukaryotic XORs, R. capsulatus XDH has an (␣)2 heterotetrameric structure, and the cofactors were identified to be located on two different polypeptides: the iron-sulfur clusters and the FAD are bound by the XdhA subunit, and the molybdenum cofactor (Moco) is bound by the XdhB subunit [15]
Biosynthesis of R. capsulatus XDH is even more complicated, requiring the assembly of an (␣)2 heterotetramer, which is a dimer of dimers
Summary
Bacterial Strains, Plasmids, Media, and Growth Conditions— E. coli TP1000(⌬mobAB) cells [20] were used for the expression of XDH wild type and variants from plasmid pSL207 [16]. For expression of chimeric (␣)2(1wt/2EB730A) XDH E. coli RK4353(DE3)mobABϪ cells cotransformed with pSL239 and pSS2 were grown in LB medium supplemented with 150 g/ml ampicillin, 50 g/ml chloramphenicol, 1 mM molybdate, and 0.02 mM isopropyl--D-thiogalactopyranoside until the A600 ϭ 1. These precultures were used 1:500 to start the main culture and subsequently grown at 30 °C until A600 ϭ 0.3 was reached. CD Spectroscopy—UV-visible CD spectra of 1.7 mg/ml enzyme samples were recorded in 50 mM Tris, 1 mM EDTA, pH 7.5, using a Jasco J-715 CD spectrophotometer. Second integrals from the simulated FeSI and FeSII spectra were used to estimate the relative amount of both clusters in the respective samples
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