Dataset Information

Synthesis of Stable NAD⁺ Mimics as Inhibitors for the Legionella pneumophila Phosphoribosyl Ubiquitylating Enzyme SdeC.

ABSTRACT: 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 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 IC₅₀ values of 28 and 39 μM, respectively.

SUBMITTER: Madern JM

PROVIDER: S-EPMC7687180 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Publications

Synthesis of Stable NAD+ Mimics as Inhibitors for the Legionella pneumophila Phosphoribosyl Ubiquitylating Enzyme SdeC.

Madern Jerre M JM Kim Robbert Q RQ Misra Mohit M Dikic Ivan I Zhang Yong Y Ovaa Huib H Codée Jeroen D C JDC Filippov Dmitri V DV van der Heden van Noort Gerbrand J GJ

Chembiochem : a European journal of chemical biology 20200616 20

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 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 dinucleo ...[more]

PMID: 32421893

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Project description:Legionella pneumophila grows intracellularly within a host membrane-bound vacuole that is formed in response to a bacterial type IV secretion system (T4SS). T4SS translocated Sde proteins promote phosphoribosyl-linked ubiquitination of endoplasmic reticulum protein Rtn4, but the role played by this modification is obscure due to lack of clear growth defects of mutants. To identify the steps in vacuole biogenesis promoted by these proteins, mutations were identified that unmasked growth defects in Δ sde strains. Mutations in the sdhA , ridL and legA3 genes aggravated the Δ sde fitness defect, resulting in disruption of the Legionella -containing vacuole (LCV) membrane within 2 hrs of bacterial contact with host cells. Depletion of Rab5B and sorting nexin 1 partially bypassed loss of Sde proteins, consistent with Sde blocking early endosome and retrograde trafficking, similar to roles previously demonstrated for SdhA and RidL proteins. Sde protein protection of LCV lysis was only observed shortly after infection, presumably because Sde proteins are inactivated by the metaeffector SidJ during the course of infection. Deletion of SidJ extended the time that Sde proteins could prevent vacuole disruption, indicating that Sde proteins are negatively regulated at the posttranslational level and are limited to protecting membrane integrity at the earliest stages of replication. Transcriptional analysis was consistent with this timing model for an early point of execution of Sde protein. Therefore, Sde proteins act as temporally-regulated vacuole guards during establishment of the replication niche, possibly by constructing a physical barrier that blocks access of disruptive host compartments early during biogenesis of the LCV.Significance statementMaintaining replication compartment integrity is critical for growth of intravacuolar pathogens within host cells. By identifying genetically redundant pathways, Legionella pneumophila Sde proteins that promote phosphoribosyl-linked ubiquitination of target eukaryotic proteins are shown to be temporally-regulated vacuole guards, preventing replication vacuole dissolution during early stages of infection. As targeting of reticulon 4 by these proteins leads to tubular endoplasmic reticulum aggregation, Sde proteins are likely to construct a barrier that blocks access of disruptive early endosomal compartments to the replication vacuole. Our study provides a new framework for how vacuole guards function to support biogenesis of the L. pneumophila replicative niche.

Project description:The bifunctional enzyme phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase (HisIE) catalyzes the second and third steps of histidine biosynthesis: pyrophosphohydrolysis of N1-(5-phospho-β-D-ribosyl)-ATP (PRATP) to N1-(5-phospho-β-D-ribosyl)-AMP (PRAMP) and pyrophosphate in the C-terminal HisE-like domain, and cyclohydrolysis of PRAMP to N-(5'-phospho-D-ribosylformimino)-5-amino-1-(5″-phospho-D-ribosyl)-4-imidazolecarboxamide (ProFAR) in the N-terminal HisI-like domain. Here we use UV-VIS spectroscopy and LC-MS to show Acinetobacter baumannii putative HisIE produces ProFAR from PRATP. Employing an assay to detect pyrophosphate and another to detect ProFAR, we established the pyrophosphohydrolase reaction rate is higher than the overall reaction rate. We produced a truncated version of the enzyme-containing only the C-terminal (HisE) domain. This truncated HisIE was catalytically active, which allowed the synthesis of PRAMP, the substrate for the cyclohydrolysis reaction. PRAMP was kinetically competent for HisIE-catalyzed ProFAR production, demonstrating PRAMP can bind the HisI-like domain from bulk water, and suggesting that the cyclohydrolase reaction is rate-limiting for the overall bifunctional enzyme. The overall kcat increased with increasing pH, while the solvent deuterium kinetic isotope effect decreased at more basic pH but was still large at pH 7.5. The lack of solvent viscosity effects on kcat and kcat/KM ruled out diffusional steps limiting the rates of substrate binding and product release. Rapid kinetics with excess PRATP demonstrated a lag time followed by a burst in ProFAR formation. These observations are consistent with a rate-limiting unimolecular step involving a proton transfer following adenine ring opening. We synthesized N1-(5-phospho-β-D-ribosyl)-ADP (PRADP), which could not be processed by HisIE. PRADP inhibited HisIE-catalyzed ProFAR formation from PRATP but not from PRAMP, suggesting that it binds to the phosphohydrolase active site while still permitting unobstructed access of PRAMP to the cyclohydrolase active site. The kinetics data are incompatible with a build-up of PRAMP in bulk solvent, indicating HisIE catalysis involves preferential channeling of PRAMP, albeit not via a protein tunnel.

Dataset Information

Synthesis of Stable NAD⁺ Mimics as Inhibitors for the Legionella pneumophila Phosphoribosyl Ubiquitylating Enzyme SdeC.

Publications

Synthesis of Stable NAD<sup>+</sup> Mimics as Inhibitors for the Legionella pneumophila Phosphoribosyl Ubiquitylating Enzyme SdeC.

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