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      Autoproteolytic Activation of ThnT Results in Structural Reorganization Necessary for Substrate Binding and Catalysis


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          cis-Autoproteolysis is a post-translational modification necessary for the function of ThnT, an enzyme involved in the biosynthesis of the β-lactam antibiotic thienamycin. This modification generates an N-terminal threonine nucleophile that is used to hydrolyze the pantetheinyl moiety of its natural substrate. We determined the crystal structure of autoactivated ThnT to 1.8 Å through X-ray crystallography. Comparison to a mutationally inactivated precursor structure revealed several large conformational rearrangements near the active site. To probe the relevance of these transitions, we designed a pantetheine-like chloromethyl ketone inactivator and co-crystallized it with ThnT. Although this class of inhibitor has been in use for several decades, the mode of inactivation had not been determined for an enzyme that uses an N-terminal nucleophile. The co-crystal structure revealed the chloromethyl ketone bound to the N-terminal nucleophile of ThnT through an ether linkage, and analysis suggests inactivation through a direct displacement mechanism. More importantly, this inactivated complex shows that three regions of ThnT that are critical to the formation of the substrate binding pocket undergo rearrangement upon autoproteolysis. Comparison of ThnT with other autoproteolytic enzymes of disparate evolutionary lineage revealed a high degree of similarity within the proenzyme active site, reflecting shared chemical constraints. However, after autoproteolysis, many enzymes, like ThnT, are observed to rearrange in order to accommodate their specific substrate. We propose that this is a general phenomenon, whereby autoprocessing systems with shared chemistry may possess similar structural features that dissipate upon rearrangement into a mature state.

          Graphical Abstract


          ► A 1.8-Å X-ray structure shows that peptide bond isomerization accompanies autoproteolysis. ► The threonine γ-methyl facilitates formation of a reactive N-terminal nucleophile. ► A halomethyl ketone inhibitor is shown to bind through an ether linkage. ► Computational modeling suggests inactivation through a direct displacement mechanism. ► Comparison with the proteasome β-subunit reveals general features of autoactivation.

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          Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs.

          A novel coronavirus has been identified as the causative agent of severe acute respiratory syndrome (SARS). The viral main proteinase (Mpro, also called 3CLpro), which controls the activities of the coronavirus replication complex, is an attractive target for therapy. We determined crystal structures for human coronavirus (strain 229E) Mpro and for an inhibitor complex of porcine coronavirus [transmissible gastroenteritis virus (TGEV)] Mpro, and we constructed a homology model for SARS coronavirus (SARS-CoV) Mpro. The structures reveal a remarkable degree of conservation of the substrate-binding sites, which is further supported by recombinant SARS-CoV Mpro-mediated cleavage of a TGEV Mpro substrate. Molecular modeling suggests that available rhinovirus 3Cpro inhibitors may be modified to make them useful for treating SARS.
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                Author and article information

                J Mol Biol
                J. Mol. Biol
                Journal of Molecular Biology
                Elsevier Ltd.
                14 June 2012
                28 September 2012
                14 June 2012
                : 422
                : 4
                : 508-518
                [1 ]Department of Biophysics, The Johns Hopkins University, Baltimore, MD 21218, USA
                [2 ]Department of Chemistry, The Johns Hopkins University, Baltimore, MD 21218, USA
                [3 ]Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
                Author notes
                [* ]Corresponding author. Department of Chemistry, The Johns Hopkins University, Baltimore, MD 21218, USA. ctownsend@ 123456jhu.edu

                A.R.B. and J.W.L. contributed equally to this work.


                Present address: J. W. Labonte, Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA; M. F. Freeman, Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, 53223 Bonn, Germany; N. T. Wright, Department of Chemistry, James Madison University, Harrisonburg, VA 22807, USA.

                Copyright © 2012 Elsevier Ltd. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                : 2 April 2012
                : 2 June 2012
                : 8 June 2012

                Molecular biology
                cmk, chloromethyl ketone,pβs, proteasome β‐subunit,ga, glycosylasparaginase,ca, cephalosporin acylase,hmk, halomethyl ketone,pdb, protein data bank,thienamycin biosynthesis,cis‐autoproteolysis,mechanism of chloromethyl ketone inhibition,n‐terminal nucleophile,x‐ray crystallography


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