The Discovery And Functional Studies Of Two Diphthamide Biosynthetic Genes

Diphthamide is a post-translationally modified histidine residue found in eukaryotic and archael elongation factor 2 (EF2). Diphtheria toxin (DT) and the Pseudomonas exotoxin A (ETA) catalyze the ADP-ribosylation reaction on the diphthamide residue of EF-2 and therefore inactivate EF-2 and stop ribosomal protein synthesis. Proposed biosynthetic pathway of diphthamide has three steps. In eukaryotes, there are five participating genes, namely DPH1 - DPH5, have been identified. The first step requires four proteins, Dph1-4, while the second step requires a single protein, Dph5. In contrast, the enzyme required for the last amidation step has remained unknown even three decades after the structure of diphthamide was revealed. I started my work on the first step modification. Yeast Dph1 and Dph2 were expressed and purified and were shown to be active in S-adenosyl methionine (SAM) cleavage, but not in the 3-amino-3-carboxypropyl (ACP) group transfer. Yeast ORF YBR246W was then studied, which was reported to be required for diphthamide biosynthesis. Our results showed that diphthine accumulates in the YBR246W deletion strain. Therefore YBR246W is required for the amidation step of diphthamide biosynthesis. Following this functional study, I discovered the diphthamide synthetase, YLR143W using yeast co-fitness analysis. We have been able to show that YLR143W catalyzes the diphthine amidation reaction in vitro. The nitrogen source of this reaction is ammonium and AMP is generated. The human orthologue ATPBD4 is also a diphthamide synthetase. Further study revealed that YBR246W's activity is required prior to the activity of YLR143W. So there should be a minimum of four steps in diphthamide biosynthetic pathway. The final part of my thesis describes our work on a human sirtuin SIRT5. Sirtuins are a family of NAD-dependent deacetylases. Our crystallography and biochemical studies showed that Sirt5 prefers to catalyze demalonylation and desuccinylation instead of deacetylation. A number of lysine succinylation sites were identified on bovine glutamate dehydrogenase (GDH) and other proteins. Sirt5 was shown to regulate succinylation and activity of carbamoyl-phosphate synthase 1 (CPS1)