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. 2000 Sep 26;97(20):10723-8.
doi: 10.1073/pnas.97.20.10723.

Crystal Structures of Bovine Milk Xanthine Dehydrogenase and Xanthine Oxidase: Structure-Based Mechanism of Conversion

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Free PMC article

Crystal Structures of Bovine Milk Xanthine Dehydrogenase and Xanthine Oxidase: Structure-Based Mechanism of Conversion

C Enroth et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (M(r), 290,000) bovine milk XDH at 2.1-A resolution and XO at 2.5-A resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.

Figures

Figure 1
Figure 1
(A) Molecular structure of the XDH dimer divided into the three major domains and two connecting loops. The two monomers have symmetry related domains in the same colors, in lighter shades for the monomer on the left and in darker shades for the monomer on the right. From N to C terminus, the domains are: iron/sulfur-center domain (residues 3–165; red), FAD domain (residues 226–531; green), and Mo-pt domain (residues 590–1,331; blue). The loop connecting the iron/sulfur domain with the FAD domain (residues 192–225) is shown in yellow, the one connecting the FAD domain with the Mo-pt domain (residues 537–589) is in brown, and the N and C termini are labeled. The FAD cofactor, the two iron/sulfur centers, the molybdopterin cofactor, and the salicylate also are included. The positions of residues discussed in the text are indicated. (B) For clarity, the arrangement of the cofactors and salicylate in one subunit of XDH are presented. The Mo ion is in green, the iron ions are in light blue, and the sulfur atoms in yellow.
Figure 2
Figure 2
Stereo representation of salicylate as bound in the Mo-pt active site of XDH plus corresponding 2FoFc electron density contoured at 1σ cutoff. Cofactor, inhibitor, the two sandwiching residues Phe 914 and Phe 1009, and Glu 1261 are labeled.
Figure 3
Figure 3
FAD- and Fe/S II-binding sites of XDH. The view is into the cleft toward the si-site of the flavin ring. Several amino acids are drawn in ball-and-stick mode: Thr 262, Glu 45, and Gly 48, whose main chain carbonyl atoms are close to the 7α- and 8α-methyl groups of the flavin ring; Phe 337 in stacking interaction with the re-side of the pyrimidine part of the flavin ring; Asp 429, whose side chain lies in plane with the flavin and only 3.6 Å from its C6 atom; Arg 426, whose side chain becomes the one closest to the flavin ring in XO.
Figure 4
Figure 4
Stereo representation of the change in conformation shown by an active site loop (Gln 423—Lys 433) on the XDH to XO transition; the green orientation represents the conformation it adopts in XDH and the red trace follows its path in XO. The positions of the side chains of Asp 429 and Arg 426 are indicated; they show dramatic shifts and are the major contributors to the change in electrostatic charge at the flavin site shown in Fig. 5. The view is similar to the one in Fig. 3.
Figure 5
Figure 5
The electrostatic environment looking down into the FAD binding site for XDH (A) and XO (B). The FAD molecule is shown in capped-cylinders representation. Electronegative regions are colored in red and electropositive regions in blue.

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