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In this study, we have determined the solution structure of Orf135 by NMR techniques. Based on inspection of the determined structure and monitoring the NMR signals when adding substrate, we have identified a substrate binding pocket. We also discuss the molecular recognition mechanism of Orf135 and detail a structure comparison with other Nudix enzymes. Molecular recognition of the 2-hydroxy-A AZD8055 is a particularly important issue. To date, structural investigations of MTH1 have provided the sole information pertaining to the molecular recognition of 2-hydroxy-A [14].
2. Materials and methods
2.1. Sample preparation and NMR experiments
Orf135 was expressed and purified as previously described [15]. Briefly, the protein was expressed as a villi GST-fusion protein in E. coli BL21 Star (DE3) (Invitrogen), and subsequently purified by GSH column chromatography. Following the removal of GST by HRV3C proteinase, Orf135 was finally purified by gel-filtration chromatography. Purified Orf135 was prepared in KH2PHO4-K2HPO4 (pH 6. cool 93% H2O/7% 2H2O buffer containing 50 mM KCl for the NMR experiments. NMR experiments were performed on a Bruker DMX500, a Bruker AVANCE 500 with cryogenic probe, a Bruker AVANCE 600 with cryogenic probe, or a Bruker DRX800 with triple axis gradient probe at 303 K. All spectra were processed using NMRPipe [16], and analyzed by Sparky [17]. The 1H, 13C and 15N assignments were obtained from standard multidimensional NMR methods [15].