Mechanism of Substrate and Inhibitor Binding of Rhodobacter capsulatus Xanthine Dehydrogenase

Abstract

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (␣␤) 2 heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6-aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (E B 232Q) to prevent substrate turnover. The hypoxanthine-and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue Glu B -232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward Arg B -310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a K i of 103.57 ؎ 18.96 nM for pterin-6-aldehyde.

Substrate Orientation and Catalytic Specificity in the Action of Xanthine Oxidase

“…The two orientations of hypoxanthine bound in the present crystal structure must be considered alternate Michaelis-Menten complexes formed prior to the onset of catalysis. Our results contrast with the structure recently reported for the desulfo form of the Rhodobacter capsulatus xanthine dehydrogenase with hypoxanthine, in that only one orientation of hypoxanthine was seen with the bacterial enzyme: that with C-2 toward the molybdenum center (27). Given the high degree of struc- o clockwise about the vertical axis from the perspective shown in A and C, respectively.…”

The First Mammalian Aldehyde Oxidase Crystal Structure

“…6, Table 2). It was suggested from the XDH crystal structure that Glu-802 and Arg-880 are essential for the correct positioning/orienting and/or activation of the substrate through the establishment of hydrogen bonds and electrostatic interactions (36), as found in all structures of XOR complexed to inhibitors or substrates (29,(37)(38)(39)(40). Glu-802 in bXDH is replaced by a valine in all AOX1 orthologs and by an alanine (Ala-807) in mAOX3.…”

Xanthine Dehydrogenase Active Site: Chiral Switching and Substrate Coordination

“…[60,61] I proffer three interpretations of this structure: (i) the anti-TS coordination condition for the oxomolybdenum : imino fragments is not valid in the case of purine substrates, (ii) the x-ray diffraction data for the protein : substrate co-crystal do not directly relate to the enzyme : substrate transition state, and (iii) the substrate is selected for a certain predominant proto-tautomeric form in the reaction course.…”