Crystal Structure of the Xanthine Oxidase-Related Aldehyde Oxido-Reductase from D. gigas

Abstract

The crystal structure of the aldehyde oxido-reductase (Mop) from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas has been determined at 2.25 A resolution by multiple isomorphous replacement and refined. The protein, a homodimer of 907 amino acid residues subunits, is a member of the xanthine oxidase family. The protein contains a molybdopterin cofactor (Mo-co) and two different [2Fe-2S] centers. It is folded into four domains of which the first two bind the iron sulfur centers and the last two are involved in Mo-co binding. Mo-co is a molybdenum molybdopterin cytosine dinucleotide. Molybdopterin forms a tricyclic system with the pterin bicycle annealed to a pyran ring. The molybdopterin dinucleotide is deeply buried in the protein. The cis-dithiolene group of the pyran ring binds the molybdenum, which is coordinated by three more (oxygen) ligands.

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo−C Interaction in a Biological System

“…The two AOR structures determined are very similar to each other as well as to the native structure 17,18 and a Higher resolution limit of the X-ray data. b OM1 and OR1 correspond to hydroxo and oxo ligands, respectively, in the EDO-and GOL-inhibited structures and to oxo ligands in the native structure.…”

“…27 The spectra at 20 K in Figure 2 show, in addition to the Mo(V) signal, the EPR signals associated with the proximal (FeS1, g 1 ) 2.023, g 2 ) 1.938, g 3 ) 1.919) and the distal (FeS2, g 1 ) 2.060, g 2 ) 1.9979, g 3 ) 1.900) iron-sulfur centers present in the structure of DgAOR. 17 These spectra have g-values and temperature dependence (not shown) similar to those observed in dithionite-reduced DgAOR (and other AORs from SRB) and show the g 1 splitting associated with the magnetic coupling between FeS1 and FeS2 (the g 1 feature of the FeS1 signal shows a splitting of ∼16 G; see Figure 2). 3 This indicates that neither the structure nor the chemical paths connecting FeS centers are affected on inhibition.…”

“…Because the structural data of DgAOR did not show the sulfido ligand coordinated to Mo, it was assumed that the enzyme crystallizes in the desulfo inactive form (Figure 1b). 17,18 A similar conclusion was obtained for the XO family member 4-hydroxybenzoylCoA-reductase (4-HBCR), for which structural data do not show evidence for the sulfido ligand, 24 although its presence was suggested by kinetic and spectroscopic data. 25 Studies on desulfo forms of distinct XO family members showed that the cyano- Top: Coordination around the molybdenum atom for the XO family of enzymes: (a) active form, (b) inactive desulfo form, and (c) structure of arsenite-inhibited DgAOR.…”

“…16 Aldehyde oxidoreductases (AOR) from sulfate-reducing bacteria (SRB) are members of the XO family of mononuclear Mo-enzymes that catalyze the two-electron oxidation of aldehydes to their respective carboxylic acids. The 3D structure of AOR from DesulfoVibrio gigas (DgAOR) was the first crystallographic structure reported for a member of this family 17 and the first to be structurally characterized at near atomic resolution (1.28 Å) (the term native structure will be used to refer to this structure). 18 In contrast to xanthine oxidoreductase 6 and quinoline 2-oxidoreductase, 19 there is no structural evidence supporting the presence of a sulfido ligand coordinated to the Mo atom in active forms of AORs from SRB.…”

Incorporation of either molybdenum or tungsten into formate dehydrogenase from Desulfovibrio alaskensis NCIMB 13491; EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases from sulfate-reducing bacteria

“…Slow-type EPR signals present almost-axial symmetry with resonances split by a (Table 1) within the typical values found for Mo(V) species giving slow-type signals, and in particular they are identical to those found in the D. gigas AOR [23]. This suggests that the structural characteristics of the molybdenum site of D. alaskensis FDH are similar to the one found in D. gigas AOR [24]. The spectra below 70 K showed the emergent Fe/S I EPR signal described above together with another rhombic signal, with all the g-values lower than 2 (see Fig.…”