| Summary: | 碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 102 === Abstract
dUTPase catalyzes the dUTP hydrolysis into dUMP and pyrophosphate. In addition to the production of the dTTP precursor, dUTPase prevents the miscorporation of dUTP into DNA by decreasing the dUTP/dTTP ratio. Due to these important roles, dUTPases are widely distributed in a large variety of organisms. Most available dUTPase structures are belong to the dUTPase-like superfamily. Almost all the members form a ring-like trimer and possess five highly conserved motifs involved in substrate binding and recognition, enzyme catalysis and structural stabilization. All the known structures of dUTPase-like members share similar motifs I-IV, but distinct motif V on C terminus. Bases on the motif V sequence, we classify the dUTPase-like members into two types. The motif V sequence of type I members is RGXXGFGST. The C terminal tail of type I members extends to the interface between the other two protomers, then each active site is constituted by all the three protomers. On the other hands, the motif V sequence of type II members is YX2-7KYXXQ. The C terminal tail of the type II members folds back to the protomer, then each active site in type II is constituted by two protomers. Sequence analysis of dUTPases through database reveals that most of archeal dUTPases do not have either type I or type II motif V. In this thesis, we have solved crystal structures from this kind of dUTPase to appoarch the possible functions of the C terminal motif V and the diverse substrate recognition mechanism.
Crystal structures of Methanosarcina mazei dUTPase (MmdUTPase) and MmdUTPase-dUTP were first refined at 1.45 Å and 1.53 Å resolutions with a trimer per asymmetry unit. MmdUTPase shares a high structural homology to type II members, which contain a 11-stranded barrel (、a、b、a、b、、a、'、、b and ) and two helices (N、). The subdomain I (SI) between 2 and 2b forms a long loop (L2). The active sites are located on the interfaces between each two protomers. Asp118 and Tyr121 are responsible for the specific binding of the deoxyribose moiety. Ala115, Trp117, Glu126 and Ser127 are for the specific binding and recognition of the uracil moiety, while Asn55, Arg58, Arg102*, Ser103*, Ser104*, Arg107*, Arg144* for the phosphate binding. The unique MmdUTPase-Trp117 interacts with urail through - stacking. In addition, MmdUTPase uses its unique Asn55 and Arg58 on L2 for the phosphate binding to compensate its lack of the conserved positively charged residues in motif V. Furthermore, ten residues in L2 form many extensive interaction networks at the trimeric interface, and hence L2 should play an important role in the trimeric formation, the protein structural stability and even in the protein structural folding.
Many archeal dUTPases display MmdUTPase-unique characteristics with the conserved trptophan in motif III, and conserved asparagine and arginine in the SI, but without conserved residues on the motif V. Then these archeal dUTPases may form a new type, the type III members. These members may be the older dUTPases with a shorter C terminal tail and functional L2 loop responsible for substrate binding and trimer formation. Many eubacteria dUTPases belong to the type II members. More residues on the C terminal tail had been for more interaction at the trimeric interface, and the positively charged and aromatic residues on motif V had been evolved for substrate binding and enzyme catalysis. dUTPases of eukaryotes, viruses and pathogens are type I members. More residues at the C terminal tail had been evolved for enzyme catalysis and trimer formation. Particularly the C-terminal fragment is extended to closely contact with the other two protomers with formation of more extensive interactions including an intermolecule sheet. The L2 loop was no longer needed and then had been deleted during evolution.
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