Identification Of The Multifunctional Activity Of AA-NADase And Binding And Inhibition Of Metal Ions Or Complexes To RecA Inteins

In this dissertation, two important enzymes---Agkistrodon acutus venom NADase(AA-NADase) and Mycobacterium tuberculosis RecA intein are studied. We identify the multicatalytic activities of AA-NADase and firstly find a novel AT(D)Pase-like activity for AA-NADase. Further studies show that small-molecule reductants inhibited the multicatalytic activities of AA-NADase by reducing the disulfide-bonds and Cu(II) ion. Self-catalytic protein splicing is a post translational process in which the intervening protein, intein, is cleaved from the precursor proteins with the concomitant ligation of flanking sequences. Metal ions are reported to inhibit the protein splicing in vitro or in vivo. We analyze the binding and inhibtion of divalent metal ion to RecA inteis. The initial screen has been performed on five metal ions (platinum, ruthenium, copper, zinc and gold) with different ligands by in vitro and in vivo screenning system. Results suggest the potential application of metal complexes on anti-tuberculosis. The details are summarized as follows.Chapter 1 provides a brief review of NADase and various methods for study the interaction of metal ions and protein. Part one is a detail introduction about three members of ADP ribosyl cyclase family Aplysia ADP ribosyl cyclase, CD38 and CD157. Part two is a summarization of ten methods including UV-vis, Fluorescence, NMR, MS, ITC, Equilibrium dialysis, CD, DLS, EXAFS and EPR.In Chapter 2, The AA-NADase has been purified using DEAE-Sephadex A-50 anion exchange chromatography and G-75 size exclusive chromatography. HPLC assay shows that AA-NADase has a unique multicatalytic activity. It is not only able to cleave the C-N glycosyl bond of NAD and NGD, but also able to cleave the P-O-P bond of ATP, ADP and AMP-PNP to produce AMP. The hydrolysis reactions of NAD, ATP and ADP catalyzed by AA-NADase are mutually competitive. ITC results show that AA-NADase binds with one AMP with high affinity. AA-NADase has so far been identified as the first unique multicatalytic enzyme with both NADase and AT(D)Pase-like activities.In chapter 3, The effects of the reduction of the disulfide-bonds and Cu(II) in AA-NADase by small-molecule reductants on its NADase and ADPase activities have been investigated by polyacrylamide gel electrophoresis, high performance liquid chromatography (HPLC), electron paramagnetic resonance spectroscopy (EPR) and isothermal titration calorimetry (ITC). The multicatalytic activities of AA-NADase are dependent on Cu2+ and disulfide-bonds. AA-NADase possesses one specific Cu2+-binding site with high affinity and at least has four weak Cu2+-binding sites. The assay of activity shows that the specific Cu2+-binding site with high affinity is essential for activity but the weak binding of Cu2+ ions inhibit the activity. Cu+ does not bind to apo-AA-NADase and can not recover the activity of apo-AA-NADase. Zn(II)-reconstituted AA-NADase or Mn(II)-reconstituted AA-NADase can reacquisition more activity than Cu(II)-reconstituted AA-NADase, but Ni(II) ion inhibits the activity of AA-NADase. GdnHCl-induced chemical denaturation and thermal denaturation illustrate that Cu(II) ion and the disulfide bonds significantly stabilize the structure of AA-NADase.Chapter 4 provides a brief introduction of protein splicing and intein. It includes the nomenclature, distribution, conserved motifs, type, three-dimensional structures, domains, identification, biological function and application of intein and the mechanism of protein splicing.In chspter 5, the interactions between metal ions and three minimized recA inteins have been studied. The stoichiometry, binding sites and binding affinities of metal ion to inteins are measured. Results show the metal coordinations have negligible influence on protein structure and the mobility restriction of key residues from metal coordination is likely the key cause of metal inhibition of intein splicing. The affinities of metal ions binding to inteins are from 10-7~10-11M and correlate to the inhibition efficiency of metal ions.Finally in chapter 6, we screen five metal ions (platinum, ruthenium, copper, zinc and gold) with different ligands using GFP-Intein fusion protein in vitro and the growth of E.coli. and M. tuberculosi. in vivo. Platinum complexes show overall better inhibitory activities than other complexes. The structure of the platinum complexes is important for efficiency of inhibition. The in vitro inhibition activity generally follows the trend of cis > monofunctional >> trans for the platinum compounds. Take the complex P-0810 for example, the IC50 for inhibition the recovering the fluorescence of GFP protein is 2.5μM. The inhibition of intein splicing using TS reporter system has the IC50 7.71μM and in ALSII reporter system IC50 18.47μM. M. tuberculosis growing assays show that the inhibition with IC50 10μM and MIC 40μM. Mechanism study indicates that the coordination of thiol group to platinum will prevent the formation of thioester intermediate and consequently block the protein splicing. The results suggest the potential application of platinum complexes on anti-tuberculosis a novel approach for the drug designing on tuberculosis chemotherapy.