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 araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni 2؉ affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90°C and pH 7.5 under the assay conditions used. Its apparent K m values for L-arabinose and D-galactose were 31 and 60 mM, respectively; the apparent V max values (at 90°C) were 41.3 U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalytic efficiencies (k cat /K m ) of the enzyme were 74.8 mM ؊1⅐ min ؊1 (L-arabinose) and 8.5 mM ؊1 ⅐ min ؊1 (D-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn 2؉ and/or Co 2؉ than in the absence of these ions. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield of 56% for 6 h at 80°C.Of the eubacteria whose genomes have been sequenced to date, Thermotoga maritima has the highest percentage of genes that are most similar to archaeal genes (20). This bacterium is also of evolutionary importance, because small-subunit rRNA phylogeny has shown that it is one of the deepest and most slowly evolving lineages of the eubacteria. Thus, it could be useful for elucidating the evolutionary relationship between thermophilic eubacteria and archaea. In addition, it metabolizes many simple and complex carbohydrates, including glucose, sucrose, starch, cellulose, and xylan (9). Thermostable enzymes from T. maritima involved in carbohydrate metabolism are very attractive for industrial applications (4,15,24).Almost 7% of the predicted coding sequences in the T. maritima genome are involved in metabolism of simple and complex sugars (20). Several genes encode proteins involved in arabinose metabolism. Among them, the araA gene encoding L-arabinose isomerase (AI) (EC 5.3.1.4), which catalyzes the conversion of L-arabinose to L-ribulose, not only is important for pentose sugar isomerization in vivo but also is very attractive for use in the bioconversion of D-galactose into D-tagatose in vitro (5, 25) (Fig. 1).The ketohexose D-tagatose has a sweetness value (92%) equivalent to that of sucrose but is poorly digested (18,32). This compound has been found to be a safe low-calorie sweetener in food products and is classified as a generally recognized as safe substance in the United States. It has also been classified as generally recognized as safe for u...

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.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O2 activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here, microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≤35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage-free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.

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Toxin-antitoxin (TA) systems are essential for bacterial persistence under stressful conditions. In particular, Mycobacterium tuberculosis express VapBC TA genes that encode the stable VapC toxin and the labile VapB antitoxin. Under normal conditions, these proteins interact to form a non-toxic TA complex, but the toxin is activated by release from the antitoxin in response to unfavorable conditions. Here, we present the crystal structure of the M. tuberculosis VapBC26 complex and show that the VapC26 toxin contains a pilus retraction protein (PilT) N-terminal (PIN) domain that is essential for ribonuclease activity and that, the VapB26 antitoxin folds into a ribbon-helix-helix DNA-binding motif at the N-terminus. The active site of VapC26 is sterically blocked by the flexible C-terminal region of VapB26. The C-terminal region of free VapB26 adopts an unfolded conformation but forms a helix upon binding to VapC26. The results of RNase activity assays show that Mg2+ and Mn2+ are essential for the ribonuclease activity of VapC26. As shown in the nuclear magnetic resonance spectra, several residues of VapB26 participate in the specific binding to the promoter region of the VapBC26 operon. In addition, toxin-mimicking peptides were designed that inhibit TA complex formation and thereby increase toxin activity, providing a novel approach to the development of new antibiotics.

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...

The araA gene encoding L-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65°C under the assay conditions used, and it required divalent cations such as Mn 2؉ , Co 2؉ , and Mg 2؉ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent K m values of the recombinant AAAI for L-arabinose and D-galactose were 48.0 mM (V max , 35.5 U/mg) and 129 mM (V max , 7.5 U/mg), respectively, at pH 6 and 65°C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (k cat /K m ) of each mutant at different pHs was significantly affected by an increase or decrease in V max . From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.