Investigation Of Colorimetric Assay For Fe(?)/2-oxoglutarate-dependent Dioxygenase And Metallocofactor Of Ribonucleotide Reductase

The Fe???/2-oxoglutarate-dependent dioxygenases?2-OGDs?catalyze the oxidation of substrates ranging from small molecules to large biomolecules with concomitant oxidation of co-substrate?2-oxoglutarate?into succinate.The important roles of 2-OGDs in biological processes are highlighted by their involvement in protein post-translational modification,lipid metabolism,oxygen sensing,DNA and RNA repair,and biosynthesis of antibiotics.Existing 2-OGDs activity assays based on the chemical transformation of 2-oxoglutarate include the employment of radioactive and/or fluorescence molecules.Radioactive assays raise the safety concern and on the other hand quenching of fluorescent signal by proteins may be problematic.In the present study,we reported a coupled colorimetric assay that can be generally applied to measure the activities of all members of 2-OGDs family.Succinyl-CoA synthetase is employed as the coupling enzyme to transform succinate produced from2-OGDs catalysis to form succinyl-CoA with concomitant hydrolysis of ATP to form ADP and orthophosphate.Orthophosphate can be quantitated by reacting it with molybdic acid forming a blue pigment.As a proof of concept,kinetic parameters of ectoine hydroxylase obtained using this method are compared to a traditional time-and labor-consuming HPLC based method.As 2-OGDs family enzymes are important drug targets due to their impressive versatility in catalyzing numerous oxidative reactions that are still very challenging using synthetic chemistry,colorimetric method detailed in this thesis has the potential to enable the practice of high throughput drug screening for 2-OGDs.Ribonucleotide reductases?RNRs?play indispensable roles in both DNA replication and repair.RNRs catalyze the reduction of ribonucleoside 5-di-or triphosphates?NDPs or NTPs?to deoxyribonucleoside 5-di-or triphosphates?dNDPs or dNTPs?.The reduction from NDPs?or NTPs?to dNDPs?or dNTPs?is the only pathway for the de novo synthesis of the monomeric building blocks of DNA.Epstein Barr virus?EBV?is a ubiquitous oncogenic herpesvirus that infects over 90%of the human population.Through sequence alignment,we have found that EBV RNR seems distinct from other reported class I RNR subclasses.Thus we investigated the nature of EBV RNR cofactor by in vitro reconstitution followed by activity assay and spectroscopic characterization.Initially we purified EBV RNR?subunit and reconstituted tyrosyl radical on?subunit by adding Fe²⁺and ascorbate to the lysate.To improve the solubility of?subunit,MBP tag containing EBV RNR?subunit was purified by one-step Ni-NTA column and then MBP tag was removed by digestion with Factor Xa.Then the holoenzyme activity assay was performed by combining?and?subunits adopting HPLC method.RNR is a proven anti-cancer and antiviral drug target.Till date many drugs targeting RNR have been developed,some of them are in clinical use and others are in clinical trial.Given the essentiality of the metal center for the activity of RNR,characterizing the EBV RNR metal cofactor and further study of its biosynthesis will provide the basis for developing antiviral drugs specifically against EBV and other viruses which contains similar architecture of metallocofactor assembly.