Abstract
Mammalian xanthine oxidoreductase can exist in both dehydrogenase and oxidase forms. Conversion between the two is implicated in such diverse processes as lactation, anti‐bacterial activity, reperfusion injury and a growing number of diseases. We have constructed a variant of the rat liver enzyme that lacks the carboxy‐terminal amino acids 1316–1331; it appears to assume an intermediate form, exhibiting a mixture of dehydrogenase and oxidase activities. The purified variant protein retained ~ 50–70% of oxidase activity even after prolonged dithiothreitol treatment, supporting a previous prediction that the C‐terminal region plays a role in the dehydrogenase to oxidase conversion. In the crystal structure of the protein variant, most of the enzyme stays in an oxidase conformation. After 15 min of incubation with a high concentration of NADH, however, the corresponding X‐ray structures showed a dehydrogenase‐type conformation. On the other hand, disulfide formation between Cys535 and Cys992, which can clearly be seen in the electron density map of the crystal structure of the variant after removal of dithiothreitol, goes in parallel with the complete conversion to oxidase, resulting in structural changes identical to those observed upon proteolytic cleavage of the linker peptide. These results indicate that the dehydrogenase–oxidase transformation occurs rather readily and the insertion of the C‐terminal peptide into the active site cavity of its subunit stabilizes the dehydrogenase form. We propose that the intermediate form can be generated (e.g. in endothelial cells) upon interaction of the C‐terminal peptide portion of the enzyme with other proteins or the cell membrane.DatabaseCoordinate sets and structure factors for the four crystal structures reported in the present study have been deposited in the Protein Data Bank under the identification numbers 4YRW, 4YTZ, 4YSW, and 4YTY.
Highlights
Eukaryotic xanthine oxidoreductase (XOR) is a homodimeric protein with a relative molecular mass of 290 000 and is composed of independent subunits; each subunit contains one molybdopterin, two nonidentical [2Fe-2S] clusters and one FAD as cofactors [1,2]
It followed the pattern shown by the wild-type enzyme, and xanthine oxidase (XO) activity increased with a concomitant decrease of xanthine dehydrogenase (XDH) activity
At the end of purification, the enzyme exhibited only XO activity and the activity-to-flavin ratio (AFR25; i.e. change in absorbance at 295 nmÁminÀ1 divided by the absorbance at 450 nm at 25 °C) values of the protein variant were in the range 100–160, indicating that 50–80% of the enzyme was in the active form [42,44,45,46,47]
Summary
Eukaryotic xanthine oxidoreductase (XOR) is a homodimeric protein with a relative molecular mass of 290 000 and is composed of independent subunits; each subunit contains one molybdopterin, two nonidentical [2Fe-2S] clusters (designated as Fe/SI and Fe/ SII; distinguished by redox potential and EPR signal) and one FAD as cofactors [1,2]. Disorder in the preceding stretch of amino acids leading to a break in electron density made it impossible to decide whether the C-terminal peptide in the binding site originated from the same subunit or from that of a neighboring molecule in the crystal lattice, the latter with high probability would be a crystal artifact [41] Such flexibility and the slower modification rate of Cys1316 and Cys1324 by chemical modifying agents [14,41] suggested that the C-terminal peptide switches between inserted into and being outside the cavity. We used site-directed mutagenesis to construct a protein variant lacking the sixteen carboxy-terminal residues (amino acids 1316–1331; CD-protein variant) to further investigate the role of this C-terminal peptide We found that this variant appears to be an intermediate form; it functions as if a mixture of XDH and XO forms. When comparing the C-terminal structures between the NADH-bound forms of both the CD protein variant and the C535A/ C992R/C1324S triple mutant, a remarkably stable XDH form, it became clear that insertion of the C-terminal peptide into the dinucleotide-binding cavity of its own subunit plays a crucial role in stabilizing the XDH form and generating a functional NAD+/ NADH binding site
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