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Title:Biochemical and structural characterization of radical SAM enzymes Elp3 and viperin
Author(s):Selvadurai, Kiruthika
Director of Research:Huang, Raven
Doctoral Committee Chair(s):Huang, Raven
Doctoral Committee Member(s):Martinis, Susan; Silverman, Scott; Tajkhorshid, Emad
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Elp3, Viperin, Radical SAM Enzymes
Abstract:The radical SAM (S-adenosylmethionine) superfamily consists of a diverse set of enzymes which use [4Fe-4S] and SAM to initiate radical transformations. These diverse enzymes undergo a common series of steps where SAM is cleaved to yield methionine and a 5’-deoxyadenosyl radical intermediate (5’-dA•), which usually abstracts a hydrogen atom from the substrate. This study characterizes unresolved biochemical functions of two radical SAM enzymes: Elp3 and viperin. The eukaryotic Elongator complex is a large protein complex consisting of six Elongator proteins (Elp1-Elp6) and has been associated with several cellular processes and neurological diseases in humans. Of the six subunits, Elp3 is considered the catalytic subunit with two putative enzymatic activities: acetyltransferase activity for histone acetylation and radical SAM dependent activity for the modification of uridine at the wobble position (U34) of cytoplasmic tRNAs. The main biochemical function responsible for the many biological roles of the Elongator complex remains controversial. Accumulating evidence in the literature indicates that tRNA U34 modification plays a more prominent role. This modification takes place in approximately 25% of cytoplasmic tRNAs in eukaryotic organisms. In contrast, mitochondrial and bacterial tRNAs experience a different U34 modification, carried out by the MnmE-MnmG heterotetramer. Bioinformatics analysis has shown that Elp3 is the only subunit that is present in most archaea, a few bacteria and two viruses. Furthermore, the genes encoding MnmE-MnmG are absent in these organisms. This indicates that the tRNA U34 modification found in archaeal and bacterial species (with Elp3) is likely the same as the one found in cytoplasmic tRNAs of eukaryotes. Since these species do not have Elp1-2 or Elp4-6, we further propose that Elp3 alone performs the modification reaction. Following chemical reconstitution of the [4Fe-4S] in Elp3 from Methanocaldococcus infernus (MinElp3), the modification reaction was successfully reconstituted inside an anaerobic chamber using synthetic tRNA. The modified base was identified as cm5U by RP-HPLC and LC-MS. Analysis of 5’-dA generated in the presence of deuterated substrates indicated that 5’-dA• abstracts a hydrogen atom from acetyl-CoA. To investigate the in vivo U34 modification, total tRNAs and tRNAArg were isolated from yeast, Methanococcus maripaludis (Euryarchaeota) and Sulfolobus acidocaldarius (Crenarchaeota). Neither cm5U nor mcm5U/ncm5U (modification in eukarya) was detected in the archaea; however, two new substances (549 Da and 454 Da) were detected. Further analysis is required to determine if these substances are the result of a secondary reaction on cm5U. The SAM domain of Elp3 contains the characteristic CX4CX2C motif which binds [4Fe-4S]. To aid in purification and crystallization, the cysteine residues in the motif were mutated to serine residues. Crystals were obtained from the (6xHis)-MinElp3 C95S/C98S mutant. Also, using the optimized crystallization conditions, small crystals of wild-type MinElp3 were obtained. The functional characterization of Elp3 reported in our publication, along with the evidence that the Elongator complex is mainly present in the cytoplasm, suggests that tRNA U34 modification is the ancient and primary biological function of the Elongator complex. The first response to viral infection in mammalian cells is the induction of the interferon system, which activates IFN-stimulated genes (ISGs) to inhibit various stages of viral replication. Viperin (for virus inhibitory protein, endoplasmic reticulum associated, interferon inducible) is one of the ISGs which has antiviral activity against a wide range of RNA and DNA viruses, however the molecular mechanism of viral inhibition remains unknown. Our studies indicate that the 5’-dA• generated by the recombinant viperin from Methanofollis liminatans (archaea), Trichoderma virens (fungi) and human adds to the carbon-carbon double bond of isopentenyl pyrophosphate (IPP) in vitro, resulting in the formation of a new compound 4-adenosyl isopentenyl pyrophosphate (AIPP). The crystal structure of the Trichoderma virens viperin revealed that the enzyme forms a central channel, where the substrate IPP and cofactor SAM meet. The studies suggest a mechanism of viperin inhibiting viral infection by depleting IPP, resulting in reduction of downstream biological processes such as cholesterol biosynthesis and protein prenylation. In addition, AIPP might also contribute to viral inhibition by potentially acting as a signaling molecule or an inhibitor of a yet to be identified target participating in anti-viral activities.
Issue Date:2016-08-19
Type:Thesis
URI:http://hdl.handle.net/2142/102874
Rights Information:Copyright 2016 Kiruthika Selvadurai
Date Available in IDEALS:2019-02-08
Date Deposited:2016-12


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