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The type III secretion system encoded in the Salmonella pathogenicity island-2 (SPI-2) gene cluster facilitates intracellular growth of nontyphoidal Salmonella by interfering with the maturation of Salmonella-containing vacuoles along the degradative pathway. SPI-2 gene products also protect Salmonella against the antimicrobial activity of reactive oxygen species (ROS) synthesized by the phagocyte NADPH oxidase 2 (NOX2). However, a potential relationship between inflammatory ROS and the activation of transcription of SPI-2 genes by intracellular Salmonella is unclear. Here, we show that ROS engendered in the innate host response stimulate SPI-2 gene transcription. We found that the expression of SPI-2 genes in Salmonella-sustaining oxidative stress conditions involves DksA, a protein otherwise known to regulate the stringent response of bacteria to nutritional stress. We also demonstrate that the J and zinc-2-oxidoreductase domains of DnaJ as well as the ATPase activity of the DnaK chaperone facilitate loading of DksA onto RNA polymerase complexed with SPI-2 promoters. Furthermore, the DksA-driven transcription of SPI-2 genes in Salmonella experiencing oxidative stress is contingent on upstream OmpR, PhoP, and SsrB signaling events that participate in the removal of nucleoid proteins while simultaneously recruiting RNA polymerase to SPI-2 promoter regions. Taken together, our results suggest the activation of SPI-2 gene transcription in Salmonella subjected to ROS produced by the respiratory burst of macrophages protects this intracellular pathogen against NOX2-mediated killing. We propose that Salmonella have co-opted inflammatory ROS to induce SPI-2-mediated protective responses against NOX2 host defenses. [ABSTRACT FROM AUTHOR]

Insect chitin deacetylases (CDAs) catalyze the removal of acetyl groups from chitin and modify this polymer during its synthesis and reorganization. CDAs are essential for insect survival and therefore represent promising targets for insecticide development. However, the structural and biochemical characteristics of insect CDAs have remained elusive. Here, we report the crystal structures of two insect CDAs from the silk moth Bombyx mori: BmCDA1, which may function in cuticle modification, and BmCDA8, which may act in modifying peritrophic membranes in the midgut. Both enzymes belong to the carbohydrate esterase 4 (CE4) family. Comparing their overall structures at 1.98-2.4 Å resolution with those from well-studied microbial CDAs, we found that two unique loop regions in BmCDA1 and BmCDA8 contribute to the distinct architecture of their substrate-binding clefts. These comparisons revealed that both BmCDA1 and BmCDA8 possess a much longer and wider substrate-binding cleft with a very open active site in the center than the microbial CDAs, including VcCDA from Vibrio cholerae and ArCE4A from Arthrobacter species AW19M34-1. Biochemical analyses indicated that BmCDA8 is an active enzyme that requires its substrates to occupy subsites 0,+1, and +2 for catalysis. In contrast, BmCDA1 also required accessory proteins for catalysis. To the best of our knowledge, our work is the first to unveil the structural and biochemical features of insect proteins belonging to the CE4 family. [ABSTRACT FROM AUTHOR]

Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate, which are critical fuel metabolites of skeletal muscle particularly during exercise. However, the physiological relevance of LDH remains poorly understood. Here we show that Ldhb expression is induced by exercise inhumanmuscle and negatively correlated with changes in intramuscular pH levels, a marker of lactate production, during isometric exercise. We found that the expression of Ldhb is regulated by exerciseinduced peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Ldhb gene promoter reporter studies demonstrated that PGC-1α activates Ldhb gene expression through multiple conserved estrogen-related receptor (ERR) and myocyte enhancer factor 2 (MEF2) binding sites. Transgenic mice overexpressing Ldhb in muscle (muscle creatine kinase (MCK)- Ldhb) exhibited increased exercise performance and enhanced oxygen consumption during exercise. MCK-Ldhb muscle was shown to have enhanced mitochondrial enzyme activity and increased mitochondrial gene expression, suggesting an adaptive oxidative muscle transformation. In addition, mitochondrial respiration capacity was increased and lactate production decreased inMCK-Ldhbskeletal myotubes in culture. Together, these results identified a previously unrecognized Ldhb-driven alteration in muscle mitochondrial function and suggested a mechanism for the adaptive metabolic response induced by exercise training. [ABSTRACT FROM AUTHOR]

7-Hydroxymethyl chlorophyll a reductase (HCAR) catalyzes the second half-reaction in chlorophyll b to chlorophyll a conversion. HCAR is required for the degradation of light-harvesting complexes and is necessary for efficient photosynthesis by balancing the chlorophyll a/b ratio. Reduction of the hydroxymethyl group uses redox cofactors [4Fe-4S] cluster and FAD to transfer electrons and is difficult because of the strong carbonoxygen bond. Here, we report the crystal structure of Arabidopsis HCAR at 2.7-Å resolution and reveal that two [4Fe-4S] clusters and one FAD within a very short distance form a consecutive electron pathway to the substrate pocket. In vitro kinetic analysis confirms the ferredoxin-dependent electron transport chain, thus supporting a proton-activated electron transfer mechanism. HCAR resembles a partial reconstruction of an archaeal F420-reducing [NiFe] hydrogenase, which suggests a common mode of efficient proton-coupled electron transfer through conserved cofactor arrangements. Furthermore, the trimeric form ofHCARprovides a biological clue of its interaction with light-harvesting complex II. [ABSTRACT FROM AUTHOR]

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by a-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an a-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown. Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems. [ABSTRACT FROM AUTHOR]

Members of glycosyltransferase family 75 (GT75) not only reversibly catalyze the autoglycosylation of a conserved arginine residue with specific NDP-sugars but also exhibit NDPpyranose mutase activity that reversibly converts specific NDP-pyranose to NDP-furanose. The latter activity provides valuable NDP-furanosyl donors for glycosyltransferases and requires a divalent cation as a cofactor instead of FAD used by UDP-D-galactopyranose mutase. However, details of the mechanism for NDP-pyranose mutase activity are not clear. Here we report the first crystal structures of GT75 family NDPpyranose mutases. The novel structures of GT75 member MtdL in complex with Mn2+ and GDP, GDP-D-glucopyranose, GDPL-fucopyranose, GDP-L-fucofuranose, respectively, combined with site-directed mutagenesis studies, reveal key residues involved in Mn2+ coordination, substrate binding, and catalytic reactions. We also provide a possible catalytic mechanism for this unique type of NDP-pyranose mutase. Taken together, our results highlight key elements of an enzyme family important for furanose biosynthesis. [ABSTRACT FROM AUTHOR]

The genus Salmonella is responsible for many illnesses in humans and other vertebrate animals. We report here that Salmonella enterica serovar Typhimurium harbors three transketolases that support the non-oxidative branch of the pentose phosphate pathway. BLAST analysis identified two genes, STM14_2885 and STM14_2886, that together encode a putative transketolase (TktC) with 46-47% similarity to the known TktA and TktB isoforms. Assessing themRNAand protein expression for each of the three transketolases, we determined that all are expressed in WT cells and regulated to varying extents by the alternative sigma factor RpoS. Enzyme assays with lysates from WTand transketolase-knockout strains established that TktA is responsible for >88% of the transketolase activity in WT cells. We purified recombinant forms of each isoenzyme to assess the kinetics for canonical transketolase reactions. TktA and TktB had comparable values for Vmax (539-1362 µM NADH consumed/s), Km (80-739 µM), and catalytic efficiency (1.02 x 108-1.06 x 109 M-1/s) for each substrate tested. The recombinant form of TktC had lower Km values (23-120 µM), whereas the Vmax (7.8-16 µM NADH consumed/s) and catalytic efficiency (5.58 x 106 to 6.07 x 108 M-1/s) were 10-100-fold lower. Using a murine model of Salmonella infection, we showed that a strain lacking all three transketolases is avirulent in C57BL/6 mice. These data provide evidence that S. Typhimurium possesses three transketolases that contribute to pathogenesis. [ABSTRACT FROM AUTHOR]

Aflatoxins are a series of highly toxic and carcinogenic secondary metabolites that are synthesized by Aspergillus species. The degradation of aflatoxin enzymes is an important regulatory mechanism which modulates mycotoxin producing. The retromer complex is responsible for the retrograde transport of specific biomolecules and the vacuolar fusion in the intracellular transport. Late endosomal-associated GTPase (Rab7) has been shown to be a downstream effector protein of the retromer complex. A deficiency in the retromer complex or Rab7 results in several cellular trafficking problems in yeast and humans, like protein abnormal accumulation. However, whether retromer dysfunction is involved in aflatoxin synthesis remains unclear. Here, we report that the core retromer complex, which comprises three vacuolar protein sortingassociated proteins (AflVps26-AflVps29-AflVps35), is essential for the development of dormant and resistant fungal forms such as conidia (asexual reproductive spore) and sclerotia (hardened fungal mycelium), as well as aflatoxin production and pathogenicity, in Aspergillus flavus. In particular, we show the AflVps26-AflVps29-AflVps35 complex is negatively correlated with aflatoxin exportation. Structural simulation, sitespecific mutagenesis, and coimmunoprecipitation experiments showed that interactions among AflVps26, AflVps29, and AflVps35 played crucial roles in the retromer complex executing its core functions. We further found an intrinsic connection between AflRab7 and the retromer involved in vesicle-vacuole fusion, which in turn affected the accumulation of aflatoxin synthesis-associated enzymes, suggesting that they work together to regulate the production of toxins. Overall, these results provide mechanistic insights that contribute to our understanding of the regulatory role of the core retromer complex in aflatoxin metabolism. [ABSTRACT FROM AUTHOR]

The Golgi enzyme UDP-GlcNAc:lysosomal enzymeN-acetylglucosamine- 1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2β2γ2 hexamer, mediates the initial step in the addition of the mannose 6-phosphate targeting signal on newly synthesized lysosomal enzymes. This tag serves to direct the lysosomal enzymes to lysosomes. A key property of GlcNAc-1-phosphotransferase is its unique ability to distinguish the 60 or so lysosomal enzymes from the numerous nonlysosomal glycoproteins with identical Asn-linked glycans. In this study, we demonstrate that the two Notch repeat modules and the DNA methyltransferase-associated protein interaction domain of the αsubunit are key components of this recognition process. Importantly, different combinations of these domains are involved in binding to individual lysosomal enzymes. This study also identifies the γ-binding site on the α subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the γ subunit is required for optimal phosphorylation. These findings serve to explain how GlcNAc-1-phosphotransferase recognizes a large number of proteins that lack a common structural motif. [ABSTRACT FROM AUTHOR]

Aberrant proliferation and activation of lung fibroblasts contribute to the initiation and progression of idiopathic pulmonary fibrosis (IPF). However, the mechanisms responsible for the proliferation and activation of fibroblasts are not fully understood. The objective of this study was to investigate the role of miR-101 in the proliferation and activation of lung fibroblasts. miR-101 expression was determined in lung tissues from patients with IPF and mice with bleomycin-induced pulmonary fibrosis. The regulation of miR-101 and cellular signaling was investigated in pulmonary fibroblasts in vitro. The role of miR-101 in pulmonary fibrosis in vivo was studied using adenovirus-mediated gene transfer in mice. The expression of miR-101 was down-regulated in fibrotic lungs from patients with IPF and bleomycin-treated mice. The down-regulation of miR-101 occurred via the E26 transformation-specific (ETS) transcription factor. miR-101 suppressed the WNT5a-induced proliferation of lung fibroblasts by inhibiting NFATc2 signaling via targeting Frizzled receptor 4/6 and the TGF-β-induced activation of lung fibroblasts by inhibition of SMAD2/3 signaling via targeting the TGF-β receptor 1. Adenovirus-mediated miR-101 gene transfer in the mouse lung attenuated bleomycin-induced lung fibrosis and improved lung function. Our data suggest that miR-101 is an anti-fibrotic microRNA and a potential therapeutic target for pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

The tetratricopeptide repeat (TPR)-containing protein FLU is a negative regulator of chlorophyll biosynthesis in plants. It directly interacts through its TPR domain with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme in the formation of δ-aminolevulinic acid (ALA). Delineation of how FLU binds to GluTR is important for understanding the molecular basis for FLU-mediated repression of synthesis of ALA, the universal tetrapyrrole precursor. Here, we characterize the FLU-GluTR interaction by solving the crystal structures of the uncomplexed TPR domain of FLU (FLUTPR) at 1.45-Å resolution and the complex of the dimeric domain of GluTR bound to FLUTPR at 2.4-Å resolution. Three non-canonical TPR motifs of each FLUTPR form a concave surface and clamp the helix bundle in the C-terminal dimeric domain of GluTR. We demonstrate that a 2:2 FLUTPR-GluTR complex is the functional unit for FLU-mediated GluTR regulation and suggest that the formation of the FLU-GluTR complex prevents glutamyl-tRNA, the GluTR substrate, from binding with this enzyme. These results also provide insights into the spatial regulation of ALA synthesis by the membrane-located FLU protein. [ABSTRACT FROM AUTHOR]

Magnesium protoporphyrin IX O-methyltransferase (ChlM) catalyzes transfer of the methyl group from S-adenosylmethionine to the carboxyl group of the C13 propionate side chain of magnesium protoporphyrin IX. This reaction is the second committed step in chlorophyll biosynthesis from protoporphyrin IX. Here we report the crystal structures of ChlM from the cyanobacterium Synechocystis sp. PCC 6803 in complex with S-adenosylmethionine and S-adenosylhomocysteine at resolutions of 1.6 and 1.7 Å, respectively. The structures illustrate the molecular basis for cofactor and substrate binding and suggest that conformational changes of the two "arm" regions may modulate binding and release of substrates/products to and from the active site. Tyr-28 and His- 139 were identified to play essential roles for methyl transfer reaction but are not indispensable for cofactor/substrate binding. Based on these structural and functional findings, a catalytic model is proposed. [ABSTRACT FROM AUTHOR]