Abstract
The human mitochondrial amidoxime reducing component (hmARC) is a molybdenum cofactor-dependent enzyme that is involved in the reduction of a diverse range of N-hydroxylated compounds of either physiological or xenobiotic origin. In this study, the use of a fusion-protein approach with T4 lysozyme (T4L) to determine the structure of this hitherto noncrystallizable enzyme by X-ray crystallography is described. A set of four different hmARC-T4L fusion proteins were designed. Two of them contained either an N-terminal or a C-terminal T4L moiety fused to hmARC, while the other two contained T4L as an internal fusion partner tethered to the hmARC enzyme between two predicted secondary-structure elements. One of these internal fusion constructs could be expressed and crystallized successfully. The hmARC-T4L crystals diffracted to 1.7 Å resolution using synchrotron radiation and belonged to space group P2(1)2(1)2(1) with one molecule in the asymmetric unit. Initial attempts to solve the structure by molecular replacement using T4L did not result in electron-density distributions that were sufficient for model building and interpretation of the hmARC moiety. However, this study emphasizes the utility of the T4L fusion-protein approach, which can be used for the crystallization and structure determination of membrane-bound proteins as well as soluble proteins.