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1.
Drug Dev Res ; 85(1): e22122, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37819020

ABSTRACT

The use of cell growth-based assays to identify inhibitory compounds is straightforward and inexpensive, but is also inherently insensitive and somewhat nonspecific. To overcome these limitations and develop a sensitive, specific cell-based assay, two different approaches were combined. To address the sensitivity limitation, different fluorescent proteins have been introduced into a bacterial expression system to serve as growth reporters. To overcome the lack of specificity, these protein reporters have been incorporated into a plasmid in which they are paired with different orthologs of an essential target enzyme, in this case l-methionine S-adenosyltransferase (MAT, AdoMet synthetase). Screening compounds that serve as specific inhibitors will reduce the growth of only a subset of strains, because these strains are identical, except for which target ortholog they carry. Screening several such strains in parallel not only reveals potential inhibitors but the strains also serve as specificity controls for one another. The present study makes use of an existing Escherichia coli strain that carries a deletion of metK, the gene for MAT. Transformation with these plasmids leads to a complemented strain that no longer requires externally supplied S-adenosylmethionine for growth, but its growth is now dependent on the activity of the introduced MAT ortholog. The resulting fluorescent strains provide a platform to screen chemical compound libraries and identify species-selective inhibitors of AdoMet synthetases. A pilot study of several chemical libraries using this platform identified new lead compounds that are ortholog-selective inhibitors of this enzyme family, some of which target the protozoal human pathogen Cryptosporidium parvum.


Subject(s)
Cryptosporidiosis , Cryptosporidium , Humans , Methionine Adenosyltransferase/genetics , Methionine Adenosyltransferase/chemistry , Methionine Adenosyltransferase/metabolism , S-Adenosylmethionine/metabolism , Pilot Projects , Cryptosporidium/metabolism , Escherichia coli/genetics
2.
Anal Chem ; 94(40): 13958-13967, 2022 Oct 11.
Article in English | MEDLINE | ID: mdl-36174068

ABSTRACT

Higher-energy collisional dissociation (HCD) of modified ribonucleosides generates characteristic and highly reproducible nucleoside-specific tandem mass spectra (MS/MS). Here, we demonstrate the capability of HCD spectra in combination with spectral matching for the semi-automated characterization of ribonucleosides. This process involved the generation of an HCD spectral library and the establishment of a mass spectral network for rapid detection with high sensitivity and specificity in a retention time-independent fashion. Systematic spectral matching analysis of the MS/MS spectra of tRNA hydrolysates from different organisms has helped us to uncover evidence for the existence of novel ribonucleoside modifications such as s2Cm and OHyW-14. Such an untargeted label-free approach has the potential to be integrated with other methods, including those that use isotope labeling, to simplify the characterization of unknown modified ribonucleosides. These findings suggest the compilation of a universal spectral network, for the characterization of known and unknown ribonucleosides, could accelerate discoveries in the epitranscriptome.


Subject(s)
Ribonucleosides , Tandem Mass Spectrometry , Isotope Labeling , Nucleosides , RNA, Transfer , Ribonucleosides/analysis , Tandem Mass Spectrometry/methods
3.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 4): 290-298, 2019 Apr 01.
Article in English | MEDLINE | ID: mdl-30950830

ABSTRACT

S-Adenosyl-L-methionine (AdoMet), the primary methyl donor in most biological methylation reactions, is produced from ATP and methionine in a multistep reaction catalyzed by AdoMet synthetase. The diversity of group-transfer reactions that involve AdoMet places this compound at a key crossroads in amino-acid, nucleic acid and lipid metabolism, and disruption of its synthesis has adverse consequences for all forms of life. The family of AdoMet synthetases is highly conserved, and structures of this enzyme have been determined from organisms ranging from bacteria to humans. Here, the structure of an AdoMet synthetase from the infectious parasite Cryptosporidium parvum has been determined as part of an effort to identify structural differences in this enzyme family that can guide the development of species-selective inhibitors. This enzyme form has a less extensive subunit interface than some previously determined structures, and contains some key structural differences from the human enzyme in an allosteric site, presenting an opportunity for the design of selective inhibitors against the AdoMet synthetase from this organism.


Subject(s)
Cryptosporidium parvum/enzymology , Methionine Adenosyltransferase/chemistry , Allosteric Regulation , Amino Acid Sequence , Crystallization , Humans , Models, Molecular , Protein Multimerization , Sequence Homology, Amino Acid , Structural Homology, Protein
4.
Microbiology (Reading) ; 163(12): 1812-1821, 2017 Dec.
Article in English | MEDLINE | ID: mdl-29111970

ABSTRACT

S-adenosyl-l-methionine (AdoMet) is an essential metabolite, playing a wide variety of metabolic roles. The enzyme that produces AdoMet from l-methionine and ATP (methionine adenosyltransferase, MAT) is thus an attractive target for anti-cancer and antimicrobial agents. It would be very useful to have a system that allows rapid identification of species-specific inhibitors of this essential enzyme. A previously generated E. coli strain, lacking MAT (∆metK) but containing a heterologous AdoMet transporter, was successfully complemented with heterologous metK genes from several bacterial pathogens, as well as with MAT genes from a fungal pathogen and Homo sapiens. The nine tested genes, which vary in both sequence and kinetic properties, all complemented strain MOB1490 well in rich medium. When these strains were grown in glucose minimal medium, growth delays or defects were observed with some specific metK genes, defects that were dramatically reduced if l-methionine was added to the medium.


Subject(s)
Escherichia coli/enzymology , Escherichia coli/metabolism , Methionine Adenosyltransferase/deficiency , S-Adenosylmethionine/metabolism , Bacteria/enzymology , Bacteria/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fungi/enzymology , Fungi/genetics , Genetic Complementation Test , Humans , Methionine/metabolism , Methionine Adenosyltransferase/genetics
5.
Protein Expr Purif ; 119: 11-8, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26550943

ABSTRACT

Canavan disease (CD) is a neurological disorder caused by an interruption in the metabolism of N-acetylaspartate (NAA). Numerous mutations have been found in the enzyme that hydrolyzes NAA, and the catalytic activity of aspartoacylase is significantly impaired in CD patients. Recent studies have also supported an important role in CD for the enzyme that catalyzes the synthesis of NAA in the brain. However, previous attempts to study this enzyme had not succeeded in obtaining a soluble, stable and active form of this membrane-associated protein. We have now utilized fusion constructs with solubilizing protein partners to obtain an active and soluble form of aspartate N-acetyltransferase. Characterization of the properties of this enzyme has set the stage for the development of selective inhibitors that can lower the elevated levels of NAA that are observed in CD patients and potentially serve as a new treatment therapy.


Subject(s)
Acetyltransferases/biosynthesis , Recombinant Fusion Proteins/chemistry , Acetyltransferases/chemistry , Acetyltransferases/genetics , Acetyltransferases/isolation & purification , Amino Acid Sequence , Brain/enzymology , Chromatography, Affinity , Conserved Sequence , Enzyme Stability , Escherichia coli , Humans , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification
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