Legionnaires' disease is caused by infection with the intracellularly replicating Gram-negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host... Show moreLegionnaires' disease is caused by infection with the intracellularly replicating Gram-negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (Ub(ADPr)) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole-linked Ub(ADPr), and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked Ub(ADPr). The inhibitory effects of modified Ub on the canonical eukaryotic E1-enzyme Uba1 are investigated and rationalized in the context of a high-resolution crystal structure reported herein. Finally, it is shown that synthetic Ub(ADPr) analogues can be used to effectively pull-down overexpressed DupA from cell lysate. Show less
Madern, J.M.; Kim, R.Q.; Misra, M.; Dikic, I.; Zhang, Y.; Ovaa, H.; ... ; Noort, G.J.V. van 2020
Stable NAD(+)analogues carrying single atom substitutions in either the furanose ring or the nicotinamide part have proven their value as inhibitors for NAD(+)-consuming enzymes. To investigate the... Show moreStable NAD(+)analogues carrying single atom substitutions in either the furanose ring or the nicotinamide part have proven their value as inhibitors for NAD(+)-consuming enzymes. To investigate the potential of such compounds to inhibit the adenosine diphosphate ribosyl (ADPr) transferase activity of the Legionella SdeC enzyme, we prepared three NAD(+)analogues, namely carbanicotinamide adenosine dinucleotide (c-NAD(+)), thionicotinamide adenosine dinucleotide (S-NAD(+)) and benzamide adenosine dinucleotide (BAD). We optimized the chemical synthesis of thionicotinamide riboside and for the first time used an enzymatic approach to convert all three ribosides into the corresponding NAD(+)mimics. We thus expanded the known scope of substrates for the NRK1/NMNAT1 enzyme combination by turning all three modified ribosides into NAD(+)analogues in a scalable manner. We then compared the three NAD(+)mimics side-by-side in a single assay for enzyme inhibition on Legionella effector enzyme SdeC. The class of SidE enzymes to which SdeC belongs was recently identified to be important in bacterial virulence, and we found SdeC to be inhibited by S-NAD(+)and BAD with IC(50)values of 28 and 39 mu M, respectively. Show less
