Abstract
In Mycobacterium tuberculosis the sulfate activating complex provides a key branching point in sulfate assimilation. The complex consists of two polypeptide chains, CysD and CysN. CysD is an ATP sulfurylase that, with the energy provided by the GTPase activity of CysN, forms adenosine-5’-phosphosulfate (APS) which can then enter the reductive branch of sulfate assimilation leading to the biosynthesis of cysteine. The CysN polypeptide chain also contains an APS kinase domain (CysC) that phosphorylates APS leading to 3’-phosphoadenosine-5’-phosphosulfate, the sulfate donor in the synthesis of sulfolipids. We have determined the crystal structures of CysC from M. tuberculosis as a binary complex with ADP, and as ternary complexes with ADP and APS and the ATP mimic AMP-PNP and APS, respectively, to resolutions of 1.5 Å, 2.1 Å and 1.7 Å, respectively. CysC shows the typical APS kinase fold, and the structures provide comprehensive views of the catalytic machinery, conserved in this enzyme family. Comparison to the structure of the human homolog show highly conserved APS and ATP binding sites, questioning the feasibility of the design of specific inhibitors of mycobacterial CysC. Residue Cys556 is part of the flexible lid region that closes off the active site upon substrate binding. Mutational analysis revealed this residue as one of the determinants controlling lid closure and hence binding of the nucleotide substrate.
Highlights
Mycobacterium tuberculosis, the agent that causes tuberculosis, is one of the most devastating human pathogens
The structure of CysC from M. tuberculosis was determined in complex with different nucleotide combinations: ADP, ADP/APS, and AMP-PNP/APS
The structures of the binary and ternary complexes provide the first insights into the enzyme-ligand interactions of APS kinase from a major human pathogen
Summary
Mycobacterium tuberculosis, the agent that causes tuberculosis, is one of the most devastating human pathogens. During the course of infection, M. tuberculosis is able to down-regulate its metabolic activity and persist for years in the PLOS ONE | DOI:10.1371/journal.pone.0121494. Crystal Structures of Mycobacterial CysC infected lung tissue until it is reactivated and exits the dormant phase [1]. One third of the world’s population is infected by dormant M. tuberculosis [2] which constitutes a threatening reservoir for new infections. Treatment of tuberculosis requires an unusually long chemotherapy, where not the least lack of patient compliance increases the occurrence of multidrug-resistant (MDR) or extremely drug-resistant (XDR) strains of M. tuberculosis. The elucidation of the molecular events and mechanisms that enable the bacterium to survive the conditions thought to prevail in granulomas during dormancy, i.e. nutrient starvation, hypoxia and oxidative and cell wall stress, is crucial to understand the disease and identify new therapeutic targets for chemotherapy
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