PREPARATION OF CLASS I AND III RIBONUCLEOTIDE REDUCTASE (RNR) AND ROLE OF THE ATP-CONE DOMAIN

Abstract

Ribonucleotide reductases (RNR) are key enzymes for transformation of RNA building blocks (NTPs) to DNA building blocks (dNTPs), an essential step in biological systems. RNRs are known to be the first enzymes identified with free radical chemistry and play an important role in having a tight control over dNTP levels, as these DNA building blocks are needed for vital cellular processes such as cell replication and DNA repair. Such a tight control is mediated through allosteric regulation by the end product (dNTPs). RNRs could be grouped into three different classes based on the type of free radical generation. In recent few years, several structural studies on these proteins from all three classes of RNR have led to a deeper insight into their catalytic mechanisms, allosteric regulation and the respective evolutionary relationships.
In first part of the work, the truncated aerobic class I RNR from Pseudomonas aeruginosa with the deletion of one ATP cone has been studied to provide complementary structural data to its full length version and illuminate the role of the ATP cone. Truncated NrdA from Pseudomonas aeruginosa was purified and used for small angle X-ray scattering (SAXS) studies and crystallization to provide insights into eventual drug discovery purposes considering its fatality. SAXS measurements of the truncated version showed a favored dimerization over tetramerization in absence of one ATP cone.
In the second part of the work, aerobic class I RNR from Chlamydia trachomatis, with three ATP cones, has been studied. Considering the fact that Chlamydia is a pathogenic organism and RNR is crucial to its survival, the enzyme could be an important drug target. Optimization of expression and purification of the enzyme was the main focus of this part which would help in functional characterization and crystallization. Getting a molecular structure could pave the way to drug discovery.
In the third and final section of this thesis, focus has been on cloning of anaerobic class III RNR from Pseudomonas aeruginosa into various vectors with solubility enhancing tags, as previous trials of purifying the protein has failed because of insoluble expression. Unfortunately cloning was not successful. Our results indicate that among the pETM41, 60, 50 vectors used for sub- cloning, none resulted in colonies.

Popular Abstract

Proteins can be classified in various groups based on their functions such as structural proteins, enzymes, antibodies and so forth. Enzymes are made of an array of amino acids folding into a special 3D structure which speed up chemical reactions. One of the most important enzymes in biological systems is ribonucleotide reductase (RNR), which is responsible for one of the most important steps in the synthesis of DNA building blocks. DNA contains the genetic material in most biological systems, therefore survival of any organism would be drastically affected by the impaired function of this enzyme in living organisms. However disrupting RNR function can sometimes have a beneficial effect, e.g. in cancer or in the inhibition of growth of bacteria that infect humans. DNA building blocks are needed for cell replication and repair, hence it is critically important for the cell to have tight control over this DNA building blocks synthesis. For instance, mutation rates will increase if the four different DNA building blocks are unbalanced relative to each other or at the wrong overall concentration, and this is where RNR becomes very important. RNRs control the level of DNA building blocks in the cell by either stimulating or inhibiting the enzyme activity. Hence, it is critically important to obtain insights into the structural properties of these enzymes to come up with various drug-design alternatives that could be used against pathogenic bacteria.
Pseudomonas aeruginosa and Chlamydia trachomatis are two bacteria known to be the cause of diseases among humans. RNRs in these bacteria could be therapeutic targets considering their vital role in the cells. Understanding the three-dimensional structures of RNRs from these bacteria is thus of great importance. In this study, large amounts of the aforementioned bacterial RNRs have been produced and purified with a view to using them for structural studies. In particular, the role of a specific part of the enzyme (ATP-cone) has been investigated. The structural and functional findings of the enzymes in this work could be a step towards eventual drug design against these bacteria.

• author: Nayeri, Niloofar
• supervisor: Rohit Kumar
• organization: Department of Chemistry
• course: KEMT30 20161
• year: 2016
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Pseudomonas aeruginosa class I and III RNR, proteinvetenskap, protein science, Ribonucleotide reductase (RNR), Chlamydia trachomatis class I RNR
• language: English
• id: 8885561
• date added to LUP: 2016-08-01 16:36:23
• date last changed: 2016-08-01 16:36:23