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ADP-ribosyl-acceptor Hydrolase 3 (ARH3): Structural and Biochemical Insights into Substrate Specificity, Metal Selectivity, and Mechanism of Catalysis

Pourfarjam, Yasin
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1623251078029899

Abstract Details

2021, PhD, University of Cincinnati, Arts and Sciences: Chemistry.
ADP-ribosylation is a unique post-translational modification (PTM) that regulates several cellular processes such as DNA damage response, chromatin structure, and cell fate. In eukaryotic cells, this PTM is predominantly catalyzed by the PARP family of proteins (also known as ARTD) which use ß-NAD+ as the substrate to attach ADP-ribose units to the target proteins on several residues such as Aspartate/Glutamate, Serine, Lysine, Arginine, Tyrosine, and Cysteine. PARP1 is the grounding member of the family and is responsible for more than 90 percent of cellular ADP-ribosylation. Like other PTMs, ADP-ribosylation is promptly reversed by the action of PAR and MAR erasers enzymes. Poly ADP-ribose glycohydrolase is the main eraser of PAR polymers but it can not removes the last ADP-ribose attached to the proteins. The follow-up function of site-specific MAR hydrolase enzymes such as TARG1, and MacroD1/D2 (Asp/Glu specificity), ARH1 (Arginine specificity), and ARH3 (Serine specificity) complete the process of deADP-ribosylation. ARH3 is a unique enzyme among others because it not only reverses MARylated marks but also capable of dePARylation, although in a significantly lower catalytic efficiency compared to PARG. The crystal structure of hARH3:ADP-ribose:Mg2+ identifies 3 unique structural elements that are the basis for substrate recognization and specificity; (1) The Adenine cap extensively interacts with Adenine moiety of ADP-ribose which in part corrects the binding alignment of substrates in the active site of ARH3. The importance of Adenine cap for PAR hydrolase activity of ARH3 is further explained by the lack of dePARylation activity in ARH1 in which such interactions are absent. (2) The di-Mg2+ catalytic center significantly enhances the enzymatic activity of ARH3. In our structure, the di-Mg2+ center directly engages with the terminal ribose” of ADP-ribose and exposes the 1”-OH (site of cleavage) moiety to solvent while 2”-OH, and 3”-OH groups are protein masked. Therefore, this observation explains the structural reason behind the specificity of ARH3 for cleaving 1”-OH and 1”-N bonds in substrates. (3) The Glu41-flap undergoes a large structural rearrangement to allow substrates to access the active site of ARH3. In chapter 3, I focused on the role of the di-Mg2+ catalytic center for the optimal function of ARH3. My ITC results show that the binding affinity of ARH3 for ADP-ribose substantially increases when Mg and Mn occupy the active site but this improvement is moderate in the presence of Ca cations. To better understand the selectivity of Mg2+ cations over Ca2+, I solved the crystal structure of ARH3:ADP-ribose:Ca2+. The structure reveals that the hydration shelf in the two metals differs and the presence of Ca is accompanied with significant structural defects in the position of terminal ribose”. Additionally, I investigated the role of two Mg (MgA, and MgB) for substrate binding and alignment. Through a series of biochemical, biophysical, and structural studies I found the while MgA is important for the correct alignment of substrates, the MgB significantly improves the binding affinity of substrates. In the final chapter (4), I presented the development of a time-resolved FRET (TR-FRET) assay to monitor the enzymatic activity of deADP-ribosylation enzymes in a convenient and heterogeneous fashion. Notably, I studied the role of the di-Mg2+ catalytic center for the efficiency of function. I found as the length of PAR polymers gets shorter, the presence of Mg cations for effective dePARylation becomes more critical. In line with these findings, the Serine MAR reversal activity of ARH3 is dramatically higher than the dePARylation function. Generally, the enzymatic activity of ARH3 substantially increases as it moves from the end of PAR polymers towards the last ADP-ribose.
In-Kwon Kim, Ph.D. (Committee Chair)
Edward Merino, Ph.D. (Committee Member)
George Stan, Ph.D. (Committee Member)
122 p.
Biochemistry
ADP-ribosylation; Poly ADP-ribose polymerase; ADP-ribosyl acceptor hydrolase 3

Recommended Citations

Citations

  • Pourfarjam, Y. (2021). ADP-ribosyl-acceptor Hydrolase 3 (ARH3): Structural and Biochemical Insights into Substrate Specificity, Metal Selectivity, and Mechanism of Catalysis [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1623251078029899

    APA Style (7th edition)

  • Pourfarjam, Yasin. ADP-ribosyl-acceptor Hydrolase 3 (ARH3): Structural and Biochemical Insights into Substrate Specificity, Metal Selectivity, and Mechanism of Catalysis. 2021. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1623251078029899.

    MLA Style (8th edition)

  • Pourfarjam, Yasin. "ADP-ribosyl-acceptor Hydrolase 3 (ARH3): Structural and Biochemical Insights into Substrate Specificity, Metal Selectivity, and Mechanism of Catalysis." Doctoral dissertation, University of Cincinnati, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1623251078029899

    Chicago Manual of Style (17th edition)

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