Diphthamide is a post-translationally modified histidine residue found in translation elongation factor 2 (eEF-2) in all eukaryotic cells. This single modification concentrates a lot of interesting chemistry and biology. This modification involves a unique C-C bond formation reaction that links the C2 of the imidazole ring and the 3-amino-3-carboxyl propyl group derived from S-adenosylmethionine (SAM). This C-C bond formation reaction is followed by a trimethylation step and an amidation step to give the final diphthamide modification. During certain bacterial infections, this modified residue (but not the unmodified histidine residue) is specifically recognized by bacterial toxins, including diphtheria toxin and Pseudomonas exotoxin A. These toxins catalyze the transfer of an adenosine diphosphate ribose (ADP-ribose) from nicotinamide adenine dinucleotide (NAD) to one of the nitrogen atoms in the imidazole ring. The ADP-ribosylation by bacterial toxins inhibits the function of eEF-2 and thus protein synthesis, leading to cell death. Based on genetic studies, the first step of the biosynthesis, the formation of the C-C bond, requires four proteins in eukaryotes, Dph1, Dph2, Dph3, and Dph4. However, how these four proteins catalyze the reaction and why so many proteins are involved in one reaction is unknown. Heterozygous deletions of dph1 and dph4 genes are associated with several human cancers, particularly ovarian and breast cancers, raising interesting questions about the biological function of diphthamide and the tumor suppression mechanism of Dph1 and Dph4. In this proposal, we aim to study how Dph1, Dph2, Dph3, and Dph4 catalyze the first step of diphthamide biosynthesis. Understanding the biosynthesis will uncover some very interesting enzymology, as predicted by our preliminary results, and provide insights into how the biosynthesis is regulated. Understanding its biosynthesis and regulation will help to understand the biological function of this modification and the tumor suppression mechanism of Dph1 and Dph4, and may ultimately lead to the development of new ways to treat or prevent cancer.

Public Health Relevance

Diphthamide is a unique post-translational modification that occurs in all eukaryotes. The biosynthesis of diphthamide requires multiple proteins, and mutations in several of them have been connected to cancer. This proposal aims to study the mechanism of diphthamide biosynthesis and the function of each protein required for the biosynthesis. Understanding the biosynthesis will provide important insight on the function of diphthamide, the regulation of diphthamide biosynthesis, and the mechanism of tumor formation in the absence of diphthamide biosynthesis, possibly leading to new ways to treat or prevent cancer.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function A Study Section (MSFA)
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Hagan, Ann A
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Cornell University
Schools of Arts and Sciences
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Dong, Min; Horitani, Masaki; Dzikovski, Boris et al. (2017) Substrate-Dependent Cleavage Site Selection by Unconventional Radical S-Adenosylmethionine Enzymes in Diphthamide Biosynthesis. J Am Chem Soc 139:5680-5683
Lin, Zhewang; Dong, Min; Zhang, Yugang et al. (2016) Cbr1 is a Dph3 reductase required for the tRNA wobble uridine modification. Nat Chem Biol 12:995-997
Dong, Min; Horitani, Masaki; Dzikovski, Boris et al. (2016) Organometallic Complex Formed by an Unconventional Radical S-Adenosylmethionine Enzyme. J Am Chem Soc 138:9755-8
Lin, Zhewang; Su, Xiaoyang; Chen, Wei et al. (2014) Dph7 catalyzes a previously unknown demethylation step in diphthamide biosynthesis. J Am Chem Soc 136:6179-82
Dong, Min; Su, Xiaoyang; Dzikovski, Boris et al. (2014) Dph3 is an electron donor for Dph1-Dph2 in the first step of eukaryotic diphthamide biosynthesis. J Am Chem Soc 136:1754-7
Su, Xiaoyang; Lin, Zhewang; Lin, Hening (2013) The biosynthesis and biological function of diphthamide. Crit Rev Biochem Mol Biol 48:515-21
Su, Xiaoyang; Chen, Wei; Lee, Wankyu et al. (2012) YBR246W is required for the third step of diphthamide biosynthesis. J Am Chem Soc 134:773-6
Su, Xiaoyang; Lin, Zhewang; Chen, Wei et al. (2012) Chemogenomic approach identified yeast YLR143W as diphthamide synthetase. Proc Natl Acad Sci U S A 109:19983-7
Zhu, Xuling; Dzikovski, Boris; Su, Xiaoyang et al. (2011) Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] enzyme required for diphthamide biosynthesis. Mol Biosyst 7:74-81
Lin, Hening (2011) S-Adenosylmethionine-dependent alkylation reactions: when are radical reactions used? Bioorg Chem 39:161-70

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