A.阿拉伯芥雙功能核酸酶結構解析及催化機制B.台灣龜殼花毒液金屬蛋白酶之結構解析與內生三肽抑制劑之抑制作用

Abstract

The dissertation is divided into two parts. The first topic is to discuss the structure of Arabidopsis thaliana AtBFN2, a gene participates in development differentiation. The A. thaliana gene At1g68290 was expressed as a C-terminal hexahistidine fusion protein AtBFN2 (EC 3.1.30.1). The bi-functional nuclease AtBFN2 from A. thaliana depends on zinc ion for cleaving single stranded DNA and RNA to yield 5'-nucleotides. It is a glycoprotein that participates in plant development and differentiation. In this study, the crystal structure of AtBFN2 shows a bound sulfate ion in the active site, at the center of the tri-nuclear cluster of zinc ions. The protein folds into a mostly α-helical structure with five short β-strands and contains four disulfide bonds. The zinc ions are coordinated to the side chains of three Asp and five His residues, two backbone atoms of Trp1, the sulfate ion, and a water molecule. An adenine base is bound adjacent to the active site and stacks with Tyr59. The core sugar residues attached to the three N-glycosylation sites of Asn91, Asn110 and Asn184 are also observed. By comparison with the nuclease P1 structure (PDB ID: 1AK0), the AtBFN2-sulfate-adenine complex model suggests a similar catalytic mechanism, in which the reaction starts with in-line attack at the phosphate by a zinc-activated water molecule. The other topic of this dissertation is to study snake-venom metalloproteinase (SVMP). The crystal structure of TM-1, a P-I class snake-venom metalloproteinase (SVMP) from the Trimeresurus mucrosquamatus venom, was determined at 1.8-A resolution. The overall structure of TM-1 is an oblate ellipsoid that contains three disulfide crosslinks, Cys119-Cys198、Cys160-Cys182、Cys162-Cys165. At active site, one zinc ion is bound to four ligands, including three conserved histidines and one water, displaying a tetrahedral geometry. The active site shows a deep S1’ substrate-binding pocket limited by the non-conserved Pro174 at the bottom. Further comparisons with other SVMPs suggest that the deep S1’ site of TM-1 correlates with its high inhibition sensitivity to the endogenous tripeptide inhibitors. Proteolytic specificity analysis revealed that TM-1 prefers substrates having a moderate-size and hydrophobic residue at the P1’ position, consistent with our structural observation.