Expansion Of The Class Ic Ribonucleotide Reductase, Conversion of A Class Ic To A Class Ia, And Subunit Swapping Between The classes
Open Access
- Author:
- Zrelak, Gordon Michael
- Graduate Program:
- Chemistry
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- May 09, 2013
- Committee Members:
- Joseph M Bollinger Jr., Thesis Advisor/Co-Advisor
- Keywords:
- Ribonucleotide Reductase
iron and manganese dependent
Tyrosyl radical - Abstract:
- The enzyme ribonucleotide reductase catalyzes the reduction of ribonucleotides to deoxyribonucleotides providing all organisms with the necessary precursors for DNA synthesis and repair. RNRs accomplish this chemically difficult reaction through the use a transient cysteine thiyl radical (C•) which abstracts a hydrogen atom (H•) from the 3'-carbon of the bound nucleotide’s ribose moiety to initiate its reduction. Class I RNRs, the focus of this study, utilize either a homodinuclear Fe2(III/III)/ or Mn2(III/III)/ tyrosyl-radical (Y•) cofactor or a heterodinuclear Mn(IV)/Fe(III) cofactor. Class I RNRs are comprised of two non-identical protein subunits, α and β. The α subunit contains the cysteine residue which is oxidized to form the C•, the site of nucleotide reduction and binding sites for effectors. The smaller β subunit assembles the metallocofactor, ~35 Å away from the site of catalysis in α, and houses the oxidizing potential. To initiate the reduction of ribonucleotides the oxidizing equivalent stored in β translocates to α to generate the C• in a reversible process. The first part of this thesis addresses the need to expand the class Ic RNR beyond the RNR from Chlamydia trachomatis (Ct) and the ability of these new RNRs to assemble Mn(IV)/Fe(III) cofactors. In expanding the class Ic RNRs we have identified a large number of putative Ic RNRs that exhibit high levels of sequence similarity to putative class Ia RNRs. Using these similar enzymes, we addressed the question of whether the primary basis for the division of class I RNRs lies within the β subunit or if the α subunit has coevolved, Based on tehi high sequence similarity between the class Ia and class Ic RNRs we demonstrated that a class Ic RNR β subunit can be the re-engineered by site-directed mutagenesis into a class Ia β subunit capable of generating a Y• upon the addition of O2 to a diferrous center. These findings further the understanding of the differences between the class Ia and class Ic RNRs.