Abstract
Isoquinoline 1-oxidoreductase (IOR) from Brevundimonas diminuta is a mononuclear molybdoenzyme of the xanthine-dehydrogenase family of proteins and catalyzes the conversion of isoquinoline to isoquinoline-1-one. Its primary sequence and behaviour, specifically in its substrate specificity and lipophilicity, differ from other members of the family. A crystal structure of the enzyme is expected to provide an explanation for these differences. This paper describes the crystallization and preliminary X-ray diffraction experiments as well as an optimized purification protocol for IOR. Crystallization of IOR was achieved using two different crystallization buffers. Streak-seeding and cross-linking were essential to obtain well diffracting crystals. Suitable cryo-conditions were found and a structure solution was obtained by molecular replacement. However, phases need to be improved in order to obtain a more interpretable electron-density map.
Highlights
The molybdopterin-dependent enzyme xanthine dehydrogenase (XDH) has been widely studied owing to its pathogenic role in postischaemia reperfusion injury, acting as a source of reactive oxygen species (Harrison, 2002), and for its involvement in gout (Monu & Pope, 2004)
The enzyme is a member of the mononuclear molybdenum family of proteins that have been grouped into three subfamilies (Hille, 1996), one of which includes XDH and related enzymes
The structure of the closely related CO dehydrogenase from Oligotropha carboxidovorans (Dobbek et al, 1999, 2002) has been solved. The latter is the only enzyme of the XDH family solved to real atomic resolution (1.1 AÊ ; Dobbek et al, 2002). The enzymes of this family consist of a large subunit or domain, which contains a mononuclear Mo at the active site that is bound to a molybdopterin cofactor through a dithiolene moiety, and a small subunit or domain containing two [2Fe±2S] clusters
Summary
The molybdopterin-dependent enzyme xanthine dehydrogenase (XDH) has been widely studied owing to its pathogenic role in postischaemia reperfusion injury, acting as a source of reactive oxygen species (Harrison, 2002), and for its involvement in gout (Monu & Pope, 2004). The latter is the only enzyme of the XDH family solved to real atomic resolution (1.1 AÊ ; Dobbek et al, 2002) The enzymes of this family consist of a large subunit or domain, which contains a mononuclear Mo at the active site that is bound to a molybdopterin cofactor through a dithiolene moiety, and a small subunit or domain containing two [2Fe±2S] clusters. IOR is a heterodimeric protein that catalyses the ®rst step of isoquinoline degradation (Fig. 1) It consists of two subunits, one of 80 kDa containing the Mo active centre and one of 16 kDa containing the two [2Fe±2S] clusters (Lehmann et al, 1994). In some cases the protein was lost during the hydrophobic interaction chromatography (HIC) step This problem was encountered previously (personal communication by SF) and depended on the cell culture used in the puri®cation. We report preliminary X-ray analysis and derivatization of the crystals using arsenite inactivation of the molybdenum active site (Boer et al, 2004)
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