Received: 1 February 2018 | Accepted: 18 May 2018 DOI: 10.1002/jcb.27169 RESEARCH ARTICLE Identification and validation of L‐asparaginase as a potential metabolic target against Mycobacterium tuberculosis Arti Kataria | Jasdeep Singh | Bishwajit Kundu Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, India Correspondence Bishwajit Kundu, Kusuma School of Biological Sciences, Block 1A, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India. Email: bkundu@bioschool.iitd.ac.in Abstract Multidrug‐resistant Mycobacterium tuberculosis (Mtb) has emerged as a major health challenge, necessitating the search for new molecular targets. A secretory amidohydrolase, L‐asparaginase of Mtb (MtA), originally implicated in nitrogen assimilation and neutralization of acidic microenvironment inside human alveolar macrophages, has been proposed as a crucial metabolic enzyme. To investigate whether this enzyme could serve as a potential drug target, it was studied for structural details and active site–specific inhibitors were tested on cultured Mycobacterial strain. The structural details of MtA obtained through comparative modeling and molecular dynamics simulations provided insights about the orchestration of an alternate reaction mechanism at the active site. This was contrary to the critical Tyr flipping mechanism reported in other asparaginases. We report the novel finding of Tyr to Val replacement in catalytic triad I along with the structural reorganization of a β‐hairpin loop upon substrate binding in MtA active site. Further, 5 MtA‐specific, active‐site–based inhibitors were obtained by following a rigorous differential screening protocol. When tested on Mycobacterium culture, 3 of these, M3 (ZINC 4740895), M26 (ZINC 33535), and doxorubicin showed promising results with inhibitory concentrations (IC 50 ) of 431, 100, and 56 μM, respectively. Based on our findings and considering stark differences with human asparaginase, we project MtA as a promising molecular target against which the selected inhibitors may be used to counteract Mtb infection effectively. KEYWORDS homology modeling, inhibitors, L‐asparaginase, molecular docking, molecular dynamics simulations, Mycobacterium, tuberculosis 1 | INTRODUCTION L‐Asparaginases belong to the amidohydrolase family of enzymes that catalyzes the conversion of L‐asparagine into L‐aspartic acid and ammonia. The antileukemic property of this enzyme has been explored comprehensively. 1-4 Moreover, for intracellular pathogens, this enzyme has been proposed as a potential molecular target. 5 L‐Asparaginase of Mycobacterium tuberculosis (Mtb), although identified and proposed as crucial for the survival of pathogen, has not yet been exploited. Unlike Escherichia coli where 2 types of asparaginases, encoded by two different genes, (cytoplasmic and periplasmic) have been reported, Mtb has a single gene that encodes a periplasmic J Cell Biochem. 2019;120:143-154. wileyonlinelibrary.com/journal/jcb © 2018 Wiley Periodicals, Inc. | 143 Arti Kataria and Jasdeep Singh contributed equally to this study.