The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis. Enhanced intracellular survival (Eis) protein, secreted by Mtb, enhances survival of Mycobacterium smegmatis (Msm) in macrophages. Mtb Eis was shown to suppress host immune defenses by negatively modulating autophagy, inflammation, and cell death through JNK-dependent inhibition of reactive oxygen species (ROS) generation. Mtb Eis was recently demonstrated to contribute to drug resistance by acetylating multiple amines of aminoglycosides. However, the mechanism of enhanced intracellular survival by Mtb Eis remains unanswered. Therefore, we have characterized both Mtb and Msm Eis proteins biochemically and structurally. We have discovered that Mtb Eis is an efficient N ɛ -acetyltransferase, rapidly acetylating Lys55 of dualspecificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7), a JNK-specific phosphatase. In contrast, Msm Eis is more efficient as an N α -acetyltransferase. We also show that Msm Eis acetylates aminoglycosides as readily as Mtb Eis. Furthermore, Mtb Eis, but not Msm Eis, inhibits LPSinduced JNK phosphorylation. This functional difference against DUSP16/MKP-7 can be understood by comparing the structures of two Eis proteins. The active site of Mtb Eis with a narrow channel seems more suitable for sequence-specific recognition of the protein substrate than the pocket-shaped active site of Msm Eis. We propose that Mtb Eis initiates the inhibition of JNK-dependent autophagy, phagosome maturation, and ROS generation by acetylating DUSP16/MKP-7. Our work thus provides insight into the mechanism of suppressing host immune responses and enhancing mycobacterial survival within macrophages by Mtb Eis.Rv2416c | lysine acetylation | antituberculosis drug N early one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb). This pathogenic bacterium causes tuberculosis, which claims the lives of millions of people every year (1). Tuberculosis has also become a global health issue owing to the increased incidences of multidrug-resistant and extensively drug-resistant strains of Mtb (2). This makes a search for targets of new antituberculosis drugs urgent. Mtb is a highly successful human pathogen, surviving and multiplying within the human macrophage cells of the infected people (3). Therefore, treatment of tuberculosis is difficult, requiring many months of taking a combination of antibiotics. Mtb has the ability to persist in the form of a long-term asymptomatic infection, referred to as latent tuberculosis (4). Latent tuberculosis becomes activated when the body's immune system is weakened. As a result, tuberculosis is the major cause of death among immuno-compromised AIDS patients (5).In mycobacterial infection, host innate immune responses may play a crucial role in early protection against Mtb infection, leading to establishment of effective adaptive immunity to tuberculosis (6). Additionally, MAPK pathways are activated by Mtb or its components and play an e...

The yeast protein Dom34 is a key component of no-go decay, by which mRNAs with translational stalls are endonucleolytically cleaved and subsequently degraded. However, the identity of the endoribonuclease is unknown. Homologs of Dom34, called Pelota, are broadly conserved in eukaryotes and archaea. To gain insights into the structure and function of Dom34/Pelota, we have determined the structure of Pelota from Thermoplasma acidophilum (Ta Pelota) and investigated the ribonuclease activity of Dom34/Pelota. The structure of Ta Pelota is tripartite, and its domain 1 has the RNA-binding Sm fold. We have discovered that Ta Pelota has a ribonuclease activity and that its domain 1 is sufficient for the catalytic activity. We also demonstrate that domain 1 of Dom34 has an endoribonuclease activity against defined RNA substrates containing a stem loop, which supports a direct catalytic role of yeast Dom34 in no-go mRNA decay.

Lipoic acid is the covalently attached cofactor of several multicomponent enzyme complexes that catalyze key metabolic reactions. Attachment of lipoic acid to the lipoyl-dependent enzymes is catalyzed by lipoate-protein ligases (LPLs). In Escherichia coli, two distinct enzymes lipoate-protein ligase A (LplA) and lipB-encoded lipoyltransferase (LipB) catalyze independent pathways for lipoylation of the target proteins. The reaction catalyzed by LplA occurs in two steps. First, LplA activates exogenously supplied lipoic acid at the expense of ATP to lipoyl-AMP. Next, it transfers the enzymebound lipoyl-AMP to the ⑀-amino group of a specific lysine residue of the lipoyl domain to give an amide linkage. To gain insight into the mechanism of action by LplA, we have determined the crystal structure of Thermoplasma acidophilum LplA in three forms: (i) the apo form; (ii) the ATP complex; and (iii) the lipoyl-AMP complex. The overall fold of LplA bears some resemblance to that of the biotinyl protein ligase module of the E. coli biotin holoenzyme synthetase/bio repressor (BirA). Lipoyl-AMP is bound deeply in the bifurcated pocket of LplA and adopts a U-shaped conformation. Only the phosphate group and part of the ribose sugar of lipoyl-AMP are accessible from the bulk solvent through a tunnel-like passage, whereas the rest of the activated intermediate is completely buried inside the active site pocket. This first view of the activated intermediate bound to LplA allowed us to propose a model of the complexes between Ta LplA and lipoyl domains, thus shedding light on the target protein/lysine residue specificity of LplA.Lipoic acid (6,8-thioctic acid or 1,2-dithiolane-3-pentanoic acid), a covalently bound cofactor, is essential for function of several key enzymes involved in oxidative metabolism in most prokaryotic and eukaryotic organisms (1). The lipoylated proteins include pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, branched-chain 2-oxoacid dehydrogenase, and the glycine cleavage system (2). In the reaction catalyzed by the lipoylating enzymes, i.e. lipoate-protein ligases (LPLs), 3 the free carboxyl group of lipoic acid is attached via an amide linkage to the ⑀-amino group of a specific lysine residue of the lipoate-accepting protein domains (termed the lipoyl domains) of these multienzyme complexes (3). The lipoamide arm protruding from a tight ␤-turn of the structure of the lipoyl domains shuttles reaction intermediates among different active sites of the multienzyme complexes (2). Although the general role of lipoic acid as the covalently attached coenzyme has been known for decades, the mechanisms by which lipoic acid is synthesized and becomes linked to its cognate proteins continue to be elucidated. In Escherichia coli two independent LPL enzymes modify lipoyl domains (4). The best characterized lipoylating enzyme is E. coli lipoate-protein ligase A (LplA). LplA utilizes exogenously supplied free lipoic acid to modify the specific lysine of the lipoyl domain. In the first step, LplA catalyzes synthesis...

Agmatine is the product of arginine decarboxylation and can be hydrolyzed by agmatinase to putrescine, the precursor for biosynthesis of higher polyamines, spermidine, and spermine. Besides being an intermediate in polyamine metabolism, recent findings indicate that agmatine may play important regulatory roles in mammals. Agmatinase is a binuclear manganese metalloenzyme and belongs to the ureohydrolase superfamily that includes arginase, formiminoglutamase, and proclavaminate amidinohydrolase. Compared with a wealth of structural information available for arginases, no threedimensional structure of agmatinase has been reported. Agmatinase from Deinococcus radiodurans, a 304-residue protein, shows ϳ33% of sequence identity to human mitochondrial agmatinase. Here we report the crystal structure of D. radiodurans agmatinase in Mn 2؉ -free, Mn 2؉ -bound, and Mn 2؉ -inhibitor-bound forms, representing the first structure of agmatinase. It reveals the conservation as well as variation in folding, oligomerization, and the active site of the ureohydrolase superfamily. D. radiodurans agmatinase exists as a compact homohexamer of 32 symmetry. Its binuclear manganese cluster is highly similar but not identical to the clusters of arginase and proclavaminate amidinohydrolase. The structure of the inhibited complex reveals that inhibition by 1,6-diaminohexane arises from the displacement of the metal-bridging water.

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IS200 transposases, present in many bacteria and Archaea, appear to be distinct from other groups of transposases. To provide a structural basis for understanding the action of IS200 transposases, we have determined the crystal structure of the SSO1474 protein from Sulfolobus solfataricus, a member of the IS200 family, in both Mn 2؉ -bound and Mn 2؉ -free forms. Its monomer fold is distinct from other classes of structurally characterized transposases. Two monomers form a tight dimer by exchanging the C-terminal ␣-helix and by merging the two central ␤-sheets into a large ␤-sheet. coming from the other monomer, is turned away from the active site, suggesting that a conformational change is likely to occur during the catalytic cycle.