Direct Detection of Products from <i>S</i>-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay

Banco, Michael T.; Mishra, Vidhi; Greeley, Samantha C; Ronning, Donald R.

doi:10.1021/acs.analchem.7b03556

Cited by 8 publications

(9 citation statements)

References 46 publications

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“…The Eab7_0806 protein was identified as a tRNA (adenine-N1-)-methyltransferase (EC 2.1.1.217). Methyltransferases are a class of enzymes very present in nature acting in the methylation of biopolymers, as proteins and small metabolites in the three domains of life [ 110 ]. These enzymes act by catalyzing a methyl donor group for a receptor molecule, which in turn generates S-adenosylmethionine (SAM-MT) and a modified methylated molecule.…”

Section: Resultsmentioning

confidence: 99%

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Functional annotation of hypothetical proteins from the Exiguobacterium antarcticum strain B7 reveals proteins involved in adaptation to extreme environments, including high arsenic resistance

et al. 2018

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Exiguobacterium antarcticum strain B7 is a psychrophilic Gram-positive bacterium that possesses enzymes that can be used for several biotechnological applications. However, many proteins from its genome are considered hypothetical proteins (HPs). These functionally unknown proteins may indicate important functions regarding the biological role of this bacterium, and the use of bioinformatics tools can assist in the biological understanding of this organism through functional annotation analysis. Thus, our study aimed to assign functions to proteins previously described as HPs, present in the genome of E. antarcticum B7. We used an extensive in silico workflow combining several bioinformatics tools for function annotation, sub-cellular localization and physicochemical characterization, three-dimensional structure determination, and protein-protein interactions. This genome contains 2772 genes, of which 765 CDS were annotated as HPs. The amino acid sequences of all HPs were submitted to our workflow and we successfully attributed function to 132 HPs. We identified 11 proteins that play important roles in the mechanisms of adaptation to adverse environments, such as flagellar biosynthesis, biofilm formation, carotenoids biosynthesis, and others. In addition, three predicted HPs are possibly related to arsenic tolerance. Through an in vitro assay, we verified that E. antarcticum B7 can grow at high concentrations of this metal. The approach used was important to precisely assign function to proteins from diverse classes and to infer relationships with proteins with functions already described in the literature. This approach aims to produce a better understanding of the mechanism by which this bacterium adapts to extreme environments and to the finding of targets with biotechnological interest.

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Section: Resultsmentioning

confidence: 99%

“…These enzymes act by catalyzing a methyl donor group for a receptor molecule, which in turn generates S-adenosylmethionine (SAM-MT) and a modified methylated molecule. The first SAM-MT discovered was catechol, a substance used in the pharmaceutical industry as an anti-cancer and anti-microbial compound [ 110 , 111 ].…”

Section: Resultsmentioning

confidence: 99%

Functional annotation of hypothetical proteins from the Exiguobacterium antarcticum strain B7 reveals proteins involved in adaptation to extreme environments, including high arsenic resistance

et al. 2018

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“…Fluorescence polarization assay has been widely implemented in high-throughput screening (HTS) and drug discovery ( Banco et al, 2018 ; Miyoshi, 2018 ; Perveen et al, 2021 ). The assay could detect the alterations in the apparent molecular weight of a fluorescent probe (or tracer) in solution by observing changes in the polarization of the sample’s emitted light.…”

Section: Resultsmentioning

confidence: 99%

Application of per-Residue Energy Decomposition to Design Peptide Inhibitors of PSD95 GK Domain

Tian

Yan

et al. 2022

Front. Mol. Biosci.

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Specific interaction between the postsynaptic density protein 95 (PSD95) and synapse-associated protein 90/postsynaptic density 95–associated protein (SAPAP) is crucial for excitatory synaptic development and plasticity. Designing inhibitors that target the guanylate kinase (GK) domain of PSD95, which is responsible for the interaction, is a promising manipulation tool for the investigation of the function of PSD95 GK and the etiology of its related psychiatric disorders. Herein, we designed new peptide inhibitors of PSD95 GK/SAPAP with higher binding affinity by using molecular dynamics simulations. First, the interactions between PSD95 GK and their reported phosphorylated and unphosphorylated peptides were explored by molecular dynamics simulations. Besides the hydrogen bonding interactions mediated by the phospho-serine (p-Ser) or corresponding phosphomimic residue Asp/Glu, the hydrophobic interactions from the other amino acids also contribute to the PSD95 GK/SAPAP interaction. As an unphosphorylated synthetic peptide with moderate binding affinity and relatively lower molecular weight, the QSF inhibitory peptide was selected for further modification. Based on per-residue energy decomposition results of the PSD95 GK/QSF complex, ten peptides were designed to enhance the binding interactions, especially the hydrophobic interactions. The top-ranked five peptides with lower binding energy were eventually synthesized. The binding affinities of the synthesized peptides were determined using fluorescence polarization (FP) assay. As expected, all peptides have higher binding affinity than the QSF peptide (Ki = 5.64 ± 0.51 μM). Among them, F10W was the most potent inhibitor (Ki = 0.75 ± 0.25 μM), suggesting that enhancement of the hydrophobic interactions is an important strategy for the design of new inhibitory peptides targeting PSD95 GK.

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“…Screening of recombinant EgtD methyltransferase activity was performed against the Cayman Chemical Kinase Inhibitor library and the histidine analogs followed the procedure previously described 13 . Briefly, stock solutions of inhibitors at a concentration of 10 mM and dissolved in 100% DMSO were added to the well of a 384-black well plate.…”

Section: Methodsmentioning

confidence: 99%

Inhibitors of Mycobacterium tuberculosis EgtD target both substrate binding sites to limit hercynine production

Sudasinghe

Banco

Ronning

2021

Sci Rep

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Ergothioneine (EGT) is a low molecular weight histidine betaine essential in all domains of life but only synthesized by selected few organisms. Synthesis of EGT by Mycobacterium tuberculosis (M. tb) is critical for maintaining bioenergetic homeostasis and protecting the bacterium from alkylating agents, oxidative stress, and anti-tubercular drugs. EgtD, an S-adenosylmethionine-dependent methyltransferase (AdoMet), catalyzes the trimethylation of L-Histidine to initiate EGT biosynthesis and this reaction has been shown to be essential for EGT production in mycobacteria and for long-term infection of murine macrophages by M. tb. In this work, library screening and structure-guided strategies identified multiple classes of M. tb EgtD inhibitors that bind in various regions of the enzyme active site. X-ray crystal structures of EgtD-inhibitor complexes confirm that L-Histidine analogs bind solely to the L-Histidine binding site while drug-like inhibitors, such as TGX-221, and S-Glycyl-H-1152 span both the L-Histidine and AdoMet binding sites. These enzyme-inhibitor complexes provide detailed structural information of compound scaffolds useful for developing more potent inhibitors that could shorten Tuberculosis treatment regimens by weakening important bacterial defenses.

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Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay

Cited by 8 publications

References 46 publications

Functional annotation of hypothetical proteins from the Exiguobacterium antarcticum strain B7 reveals proteins involved in adaptation to extreme environments, including high arsenic resistance

Functional annotation of hypothetical proteins from the Exiguobacterium antarcticum strain B7 reveals proteins involved in adaptation to extreme environments, including high arsenic resistance

Application of per-Residue Energy Decomposition to Design Peptide Inhibitors of PSD95 GK Domain

Inhibitors of Mycobacterium tuberculosis EgtD target both substrate binding sites to limit hercynine production

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