Academic Editor: Kentaro Kato Received: 5 February 2025 Revised: 7 March 2025 Accepted: 10 March 2025 Published: 13 March 2025 Citation: Chakrabarty, A.; Dutta, D.; Baidya, M.; Dutta, A.; Das, A.K.; Ghosh, S.K. Metronidazole Activation by a Deeply Entangled Dimeric Malic Enzyme in Entamoeba histolytica. Pathogens 2025, 14, 277. https:// doi.org/10.3390/pathogens14030277 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Metronidazole Activation by a Deeply Entangled Dimeric Malic Enzyme in Entamoeba histolytica Arindam Chakrabarty 1,† , Debajyoti Dutta 2,† , Mithu Baidya 3 , Anirudha Dutta 4 , Amit Kumar Das 1, * and Sudip K. Ghosh 1, * 1 Department of Bioscience and Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India; arindamc82@gmail.com 2 Department of Biotechnology, Thapar Institute of Engineering and Technology, Patiala 147004, India; debajyoti.dutta@thapar.edu 3 Department of Biosciences and Bioengineering, Indian Institute of Technology Jammu, Jammu and Kashmir 181221, India; mithu.baidya@iitjammu.ac.in 4 Department of Biological Sciences, Adamas University, Kolkata 700126, India; anirudha1.dutta@adamasuniversity.ac.in * Correspondence: amitk@bt.iitkgp.ac.in (A.K.D.); sudip@bt.iitkgp.ac.in (S.K.G.); Tel.: +91-3222-283756 (A.K.D.); +91-3222-283768 (S.K.G.) † These authors contributed equally to this work. Abstract: Metronidazole is the preferred drug for treating amoebiasis caused by Entamoeba histolytica. Its antiamoebic activity is primarily attributed to activation by various reductases. This study reports an alternative activation pathway in E. histolytica mediated by the decarboxylating malic enzyme. Functional characterization of this NADPH-dependent enzyme reveals that it is secreted into the extracellular milieu and may play a role in E. histolytica adhesion to human enteric cells. Structural analysis of the E. histolytica malic enzyme (EhME) demonstrates that the protein forms a strict dimer, with the protomers interlocked by a unique knot structure formed by two polypeptide chains. This distinctive structural feature closely aligns EhME with its prokaryotic counterparts. In conclusion, our findings reveal that E. histolytica harbors a deeply entangled dimeric malic enzyme that contributes to metronidazole susceptibility, sharing structural similarities with bacterial malic enzymes. Keywords: Entamoeba histolytica; metronidazole; crystal structure; entangled protein; knot; protozoa 1. Introduction Entamoeba histolytica (Eh), the causative agent of amoebiasis, is a leading protozoan parasite responsible for significant mortality in developing countries [1]. Metronidazole (Mtz) remains the most widely used therapeutic agent against amoebiasis. As a pro- drug, Mtz is reported to be activated in Entamoeba by pyruvate ferredoxin oxidoreductase (PFOR), ferredoxin, thioredoxin reductase, and nitroreductases [2,3]. Interestingly, in Trichomonas vaginalis (Tv), a closely related protozoan, studies have shown that even after complete loss of PFOR, cells remain susceptible to higher concentrations of Mtz [4]. How- ever, the development of Mtz-resistant Tv strains was observed after the complete loss of malic enzyme (ME) expression [5]. This finding led to the proposal of an alternative path- way, where a malic enzyme (ME) was proposed to provide electrons for Mtz activation in the absence of PFOR. In this alternative pathway, the oxidative decarboxylation of malate to Pathogens 2025, 14, 277 https://doi.org/10.3390/pathogens14030277