This A1 competitive renewal of GM088276 is a multidisciplinary collaboration to investigate the biosynthesis of diphthamide, a unique protein posttranslational modification that occurs on archaeal and eukaryotic translation elongation factor 2. This modification has been known for over 30 years and is the target of several bacterial toxins, including diphtheria toxin. However, the biosynthesis and biological function are still poorly understood. Previous studies by others suggest that there are five proteins (Dph1-5) required for the first two steps of the biosynthesis, while no proteins were identified for the thid (and last) amidation step. Interestingly, deletion of several of the biosynthesis genes is found in tumors. With previous grant support, we made a number of important findings, including the discovery of a novel radical SAM enzyme for the first step of diphthamide biosynthesis and the identification of two new proteins required for the last step of the biosynthesis. This renewal wil build on these finding to further understand the chemistry and enzymology of diphthamide biosynthesis and to elucidate the complete biosynthetic pathway for the first time. The interesting Fe-S enzyme chemistry that will be elucidated in this proposal will significantly expand the chemistry scope and mechanistic understanding of Fe-S enzymes. The understanding of diphthamide biosynthesis will help to understand the biological functions of diphthamide and why deletion of diphthamide biosynthesis genes promotes tumorigenesis.

Public Health Relevance

This proposal aims to study the biosynthesis of diphthamide as a way to understand its biological function and the mechanism of tumor formation in the absence of diphthamide biosynthesis. The proposed studies not only can lead to better understandings of novel enzymes, but may also lead to new ways to treat or prevent cancer.

National Institute of Health (NIH)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Gerratana, Barbara
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Cornell University
Schools of Arts and Sciences
United States
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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
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
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
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
Zhang, Yang; Zhu, Xuling; Torelli, Andrew T et al. (2010) Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme. Nature 465:891-6
Zhu, Xuling; Kim, Jungwoo; Su, Xiaoyang et al. (2010) Reconstitution of diphthine synthase activity in vitro. Biochemistry 49:9649-57