{"title":"Biochemical and biophysical characterization of Leishmania donovani citrate synthase.","authors":"Preeti Ranjan, Manash Sarma, Vikash Kumar Dubey","doi":"10.1016/j.ijbiomac.2024.135400","DOIUrl":null,"url":null,"abstract":"<p><p>Citrate synthase is a crucial enzyme in the TCA cycle and represents a potential therapeutic target. However, knowledge about this enzyme in Leishmania parasites remains limited. In this study, we have successfully cloned, expressed, and purified citrate synthase from Leishmania donovani (LdCS) using a bacterial system, and characterized it through various biophysical and biochemical methods. Circular dichroism analysis at physiological pH indicates that LdCS is properly folded. Further investigation into its tertiary structure using a quencher reveals that most tryptophan residues are located within the protein's hydrophobic core. Biochemical assays show that the recombinant enzyme is catalytically active, with optimal activity at pH 7.0. Kinetic studies provided parameters such as Km and Vmax. Enzyme inhibition assays revealed that LdCS activity is competitively inhibited by FDA-approved compounds-Abemaciclib, Bazedoxifene, Vorapaxar, and Imatinib-with Ki values ranging from 2 to 3 μM, demonstrating significant binding affinity. This research paves the way for exploring LdCS as a potential drug target for treating leishmaniasis.</p>","PeriodicalId":333,"journal":{"name":"International Journal of Biological Macromolecules","volume":null,"pages":null},"PeriodicalIF":7.7000,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"International Journal of Biological Macromolecules","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1016/j.ijbiomac.2024.135400","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/9/6 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Citrate synthase is a crucial enzyme in the TCA cycle and represents a potential therapeutic target. However, knowledge about this enzyme in Leishmania parasites remains limited. In this study, we have successfully cloned, expressed, and purified citrate synthase from Leishmania donovani (LdCS) using a bacterial system, and characterized it through various biophysical and biochemical methods. Circular dichroism analysis at physiological pH indicates that LdCS is properly folded. Further investigation into its tertiary structure using a quencher reveals that most tryptophan residues are located within the protein's hydrophobic core. Biochemical assays show that the recombinant enzyme is catalytically active, with optimal activity at pH 7.0. Kinetic studies provided parameters such as Km and Vmax. Enzyme inhibition assays revealed that LdCS activity is competitively inhibited by FDA-approved compounds-Abemaciclib, Bazedoxifene, Vorapaxar, and Imatinib-with Ki values ranging from 2 to 3 μM, demonstrating significant binding affinity. This research paves the way for exploring LdCS as a potential drug target for treating leishmaniasis.
期刊介绍:
The International Journal of Biological Macromolecules is a well-established international journal dedicated to research on the chemical and biological aspects of natural macromolecules. Focusing on proteins, macromolecular carbohydrates, glycoproteins, proteoglycans, lignins, biological poly-acids, and nucleic acids, the journal presents the latest findings in molecular structure, properties, biological activities, interactions, modifications, and functional properties. Papers must offer new and novel insights, encompassing related model systems, structural conformational studies, theoretical developments, and analytical techniques. Each paper is required to primarily focus on at least one named biological macromolecule, reflected in the title, abstract, and text.