The Enzymatic Mechanism Studies Of Yeast ω-Amidase Nit2and The Structural And Functional Research Of Yeast Histone Chaperone Hif1

Nit protein family, as the branch10of nitrilase superfamily, is widely distributed in animals, plants, fungi and prokaryotic cells. Mammals have two Nit proteins, namely Nitl and Nit2, which have highly conserved amino acid sequences. Yeast Nit2is a homologue of mammalian tumor-suppressor protein Nitl whose substrate specificity is not yet known. Previous studies have shown that mammalian Nit2, a putative tumor-suppressor, is identical to be an ω-amidase, an enzyme that catalyszes the specific hydrolysis of a-ketoglutaramate and a-ketosuccinamate. However, the enzymatic mechanism of mammalian Nit2is ambiguous while the natural substrates of mammalian Nitl haven’t been found out. Here, we solved crystal structures of yeast Nit2in complex with a-ketoglutarate and oxaloacetate, which were thought to be the reaction intermediates of the hydrolysis, respectively. These structures reveal that Arg173, Thr196and Thr199of yeast Nit2could bind the a-moiety of the intermediate and the highly conserved canonical catalytic triad CEK could bind the γ/β-group. In addition, highly conserved Phe195of yeast Nit2interacts with a-moiety via a newly founded η6type anion enhanced π-π interaction. Structural analysis indicates that the dimerization of yeast Nit2is important to the putative catalysis process. β6/7-Hairpin lid may trigger the catalytic process via the dimer interface. The catalytic process should be a general mechanism of Nit proteins because of the highly conserved residues involved in the catalysis and dimerization. However, the active site of yeast Nit2has important differences comparing to mammalian Nit2, resulting in extremely low specific activity towards a-ketoglutaramate, which is similar to mammalian Nitl. The volume of the binding pocket of yeast Nit2is big enough for large molecular binding. We have solved the crystal structure of yeast inactive Nit2C169S mutant protein in complex with an unknown endogenous ligand. The structure reveals that the natural substrate of yeast Nit2may be a kind of dipeptide-N-substituted analogues of a-ketoglutaramate or other similar molecules. Mammalian Nitl may have similar pocket property comparing to yeast Nit2, which means they may have similar substrate specificity. Our study of yeast Nit2would not only expend our knowledge of the catalytic mechanism of mammalian Nit2, but provide a clue for exploring the natural substrate of mammalian Nitl then understanding its biological role in the cell. Yeast Hifl was first found to be a nonessential component of nuclear B-type histone acetyltransferase complex (HAT-B). It participates in telomeric silence and promotes the precipitate of nucleosome, although it has no effect on the specific activity of HAT-B complex. In present view, as a histone chaperone protein, Hifl is essential as protein Asfl that recruits histone binding protein to the chromatin in the process of replication-coupled or-independent chromatin reassembly. In addition, nuclear autoantigenic sperm protein (NASP), human homologue of yeast Hifl, involves in many vital important life regulation progress such as cell proliferation. For the importance of Hifl, we solved its crystal structure. The structural analysis of Hifl reveals that it consists of a TPR domain and an interrupted acid loop. The TPR domain consisting of4TPRs constructs the basic structural framework of Hif1, while the interrupted acid loop binds the TPR domain and may change the conformation of the TPR skeleton. In this study, we found that Hifl could bind to histone octamer via binding to H3/H4tetramer. The acid loop is important for this kind of binding. In addition, structure-based alignment of Hifl indicates that the cave shaped by TPR domain may bind with specific peptides. According to the molecular docking experiment, the cave of TPR domain could be inosculated by histone H3tail, while the acid loop may recognize the post-translational modification state of H4tail. The crystal structure of Hifl, the first structure of SHNi-TPR family, provides insight into the structural characterization of SHNi-TPR proteins. Our research of yeast Hifl provides a preliminary structural model for our further study of the interaction between HAT-B complex and histone complex.