| Schizosaccharomyces pombe Aps1 is a nudix hydrolase that catalyzes the hydrolysis of both diadenosine 5',5'''-P1,P n-oligophosphates and diphosphoinositol polyphosphates in vitro. Nudix hydrolases, in general, act upon a wide variety of substrates, despite having a common 23 amino acid catalytic motif, hence, the residues responsible for substrate specificity for each enzyme are considered to reside outside the common catalytic nudix motif. The specific residues involved in binding each substrate of S. pombe Aps1 are unknown. The major goal of this research was to determine the structure of Aps1 in order to identify the specific residues required for binding each class of substrates. To this end, we demonstrated several major findings with regard to Aps1.;First, values of the kinetic parameters of Km and kcat determined for two N-terminal truncated and full-length forms of Aps1, by substrate saturation assays, indicated that the initial 31 residues of Aps1 are not involved in binding or hydrolysis of Ap6A. In addition, our results suggest that residues 20-31 may potentially be necessary for structural stability, since Aps1Delta2-31 was prone to precipitation as compared to the 2-19 truncation mutant.;Second, we determined the secondary structure and backbone dynamics of Aps1Delta2-19 with NMR spectroscopy. The secondary structure of Aps1Delta2-19 exhibited a combination of alpha-helices, beta-strands, and a loop-helix-loop motif, which are characteristics of the nudix hydrolase family. The backbone relaxation data indicated the presence of three dynamic loop-regions in Aps1Delta2-19. Interestingly, one of the large dynamic loop-regions of Aps1Delta2-19 corresponds to an apparent sequence insert in Aps1 unique among other putative fungal orthologs.;Third, two structural models of Aps1Delta2-19, based on the crystal structures of human DIPP1 and T. thermophilus Ndx1, were generated using the HHpred homology modeling server. The structural models were in excellent agreement with the NMR-derived secondary structure of Aps1Delta2-19. In addition, dynamic regions identified in Aps1Delta2-19 were in agreement with loop regions identified in the structural models of Aps1Delta2-19.;Fourth, NMR chemical shift mapping in conjunction with structural homology models indicated several residues outside the catalytic nudix motif that are involved in specific binding of diphosphoinositol polyphosphate or diadenosine oligophosphate ligands.;The results presented in this dissertation research enhance our understanding of the versatility of Aps1 by elucidating residues involved in binding two distinct classes of substrates. The results presented here should facilitate further studies of the in vivo function of Aps1 and other members of this subfamily of nudix hydrolases. |
