Targeting the role of a key conserved motif for substrate selection and catalysis by 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between three-carbon phosphoenolpyruvate (PEP) and five-carbon d-arabinose 5-phosphate (A5P), generating KDO8P, a key intermediate in the biosynthetic pathway to 3-deoxy-D-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Both metal-dependent and metal-independent forms of KDO8PS have been characterized. KDO8PS is evolutionarily and mechanistically related to the first enzyme of the shikimate pathway, the obligately divalent metal ion-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) that couples PEP and four-carbon D-erythrose 4-phosphate (E4P) to give DAH7P. In KDO8PS, an absolutely conserved KANRS motif forms part of the A5P binding site, whereas in DAH7PS, an absolutely conserved KPR(S/T) motif accommodates E4P. Here, we have characterized four mutants of this motif (AANRS, KAARS, KARS, and KPRS) in metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and metal-independent KDO8PS from Neisseria meningitidis to test the roles of the universal Lys and the Ala-Asn portion of the KANRS motif. The X-ray structures, determined for the N. meningitidis KDO8PS mutants, indicated no gross structural penalty resulting from mutation, but the subtle changes observed in the active sites of these mutant proteins correlated with their altered catalytic function. (1) The AANRS mutations destroyed catalytic activity. (2) The KAARS mutations lowered substrate selectivity, as well as activity. (3) Replacing KANRS with KARS or KPRS destroyed KDO8PS activity but did not produce a functional DAH7PS. Thus, Lys is critical to catalysis, and other changes are necessary to switch substrate specificity for both the metal-independent and metal-dependent forms of these enzymes.     

Probing domain interactions in soluble guanylate cyclase

Eukaryotic nitric oxide (NO) signaling involves modulation of cyclic GMP (cGMP) levels through activation of the soluble isoform of guanylate cyclase (sGC). sGC is a heterodimeric hemoprotein that contains a Heme-Nitric oxide and OXygen binding (H-NOX) domain, a Per/ARNT/Sim (PAS) domain, a coiled-coil (CC) domain, and a catalytic domain. To evaluate the role of these domains in regulating the ligand binding properties of the heme cofactor of NO-sensitive sGC, we constructed chimeras by swapping the rat β1 H-NOX domain with the homologous region of H-NOX domain-containing proteins from Thermoanaerobacter tengcongensis, Vibrio cholerae, and Caenorhabditis elegans (TtTar4H, VCA0720, and Gcy-33, respectively). Characterization of ligand binding by electronic absorption and resonance Raman spectroscopy indicates that the other rat sGC domains influence the bacterial and worm H-NOX domains. Analysis of cGMP production in these proteins reveals that the chimeras containing bacterial H-NOX domains exhibit guanylate cyclase activity, but this activity is not influenced by gaseous ligand binding to the heme cofactor. The rat-worm chimera containing the atypical sGC Gcy-33 H-NOX domain was weakly activated by NO, CO, and O(2), suggesting that atypical guanylate cyclases and NO-sensitive guanylate cyclases have a common molecular mechanism for enzyme activation. To probe the influence of the other sGC domains on the mammalian sGC heme environment, we generated heme pocket mutants (Pro118Ala and Ile145Tyr) in the β1 H-NOX construct (residues 1-194), the β1 H-NOX-PAS-CC construct (residues 1-385), and the full-length α1β1 sGC heterodimer (β1 residues 1-619). Spectroscopic characterization of these proteins shows that interdomain communication modulates the coordination state of the heme-NO complex and the heme oxidation rate. Taken together, these findings have important implications for the allosteric mechanism of regulation within H-NOX domain-containing proteins.     

The metalloprotease of Listeria monocytogenes is regulated by pH

Listeria monocytogenes is an intracytosolic bacterial pathogen. Among the factors contributing to escape from vacuoles are a phosphatidylcholine phospholipase C (PC-PLC) and a metalloprotease (Mpl). Both enzymes are translocated across the bacterial membrane as inactive proproteins, whose propeptides serve in part to maintain them in association with the bacterium. We have shown that PC-PLC maturation is regulated by Mpl and pH and that Mpl maturation occurs by autocatalysis. In this study, we tested the hypothesis that Mpl activity is pH regulated. To synchronize the effect of pH on bacteria, the cytosolic pH of infected cells was manipulated immediately after radiolabeling de novo-synthesized bacterial proteins. Immunoprecipitation of secreted Mpl from host cell lysates revealed the presence of the propeptide and catalytic domain in samples treated at pH 6.5 but not at pH 7.3. The zymogen was present in small amounts under all conditions. Since proteases often remain associated with their respective propeptide following autocatalysis, we aimed at determining whether pH regulates autocatalysis or secretion of the processed enzyme. For this purpose, we used an Mpl construct that contains a Flag tag at the N terminus of its catalytic domain and antibodies that can distinguish N-terminal and non-N-terminal Flag. By fluorescence microscopy, we observed the Mpl zymogen associated with the bacterium at physiological pH but not following acidification. Mature Mpl was not detected in association with the bacterium at either pH. Using purified proteins, we determined that processing of the PC-PLC propeptide by mature Mpl is also pH sensitive. These results indicate that pH regulates the activity of Mpl on itself and on PC-PLC.     

Genome sequences of Salmonella enterica serovar typhimurium, Choleraesuis, Dublin, and Gallinarum strains of well- defined virulence in food-producing animals

Salmonella enterica is an animal and zoonotic pathogen of worldwide importance and may be classified into serovars differing in virulence and host range. We sequenced and annotated the genomes of serovar Typhimurium, Choleraesuis, Dublin, and Gallinarum strains of defined virulence in each of three food-producing animal hosts. This provides valuable measures of intraserovar diversity and opportunities to formally link genotypes to phenotypes in target animals.     

Long-term alteration of anxiolytic effects of ovarian hormones in female mice by a peripubertal immune challenge

Recent reports indicate that exposure to some stressors, such as shipping or immune challenge with the bacterial endotoxin, lipopolysaccharide (LPS), during the peripubertal period reduces sexual receptivity in response to ovarian hormones in adulthood. We hypothesized that a peripubertal immune challenge would also disrupt the response of a non-reproductive behavior, anxiety-like behavior, to ovarian hormones in adulthood. Female C57Bl/6 mice were injected with LPS during the peripubertal period and tested for anxiety-like behavior in adulthood, following ovariectomy and ovarian hormone treatment. Treatment with estradiol followed by progesterone reduced anxiety-like behavior in control, but not LPS-treated females. We next determined if the disruptive effect of LPS on adult behavior were limited to the peripubertal period by treating mice with LPS either during this period or in adulthood. LPS treatment during the peripubertal period disrupted the anxiolytic effect of ovarian hormones, whereas treatment in adulthood did not. We further tested if this model of peripubertal immune challenge was applicable to an outbred strain of mice (CD-1). Similar to C57Bl/6 mice, LPS treatment during the peripubertal period, but not later, disrupted the anxiolytic effect of estradiol and progesterone. These data suggest that a peripubertal immune challenge disrupts the regulation of anxiety-like behavior by ovarian hormones in a manner that persists at least for weeks after the termination of the immune challenge.     

Stiffness of photocrosslinked RGD-alginate gels regulates adipose progenitor cell behavior

Adipose progenitor cells (APCs) are widely investigated for soft tissue reconstruction following tumor resection; however, the long-term success of current approaches is still limited. In order to develop clinically relevant therapies, a better understanding of the role of cell-microenvironment interactions in adipose tissue regeneration is essential. In particular, the effect of extracellular matrix (ECM) mechanics on the regenerative capability of APCs remains to be clarified. We have used artificial ECMs based on photocrosslinkable RGD-alginate to investigate the adipogenic and pro-angiogenic potential of 3T3-L1 preadipocytes as a function of matrix stiffness. These hydrogels allowed us to decouple matrix stiffness from changes in adhesion peptide density or extracellular Ca(2+) concentration and provided a physiologically relevant 3D culture context. Our findings suggest that increased matrix rigidity promotes APC self-renewal and angiogenic capacity, whereas, it inhibits adipose differentiation. Collectively, this study advances our understanding of the role of ECM mechanics in adipose tissue formation and vascularization and will aid in the design of efficacious biomaterial scaffolds for adipose tissue engineering applications.     

Evaluation of nanoparticle-immobilized cellulase for improved ethanol yield in simultaneous saccharification and fermentation reactions

Ethanol yields were 2.1 (P = 0.06) to 2.3 (P = 0.01) times higher in simultaneous saccharification and fermentation (SSF) reactions of microcrystalline cellulose when cellulase was physisorbed on silica nanoparticles compared to enzyme in solution. In SSF reactions, cellulose is hydrolyzed to glucose by cellulase while yeast simultaneously ferments glucose to ethanol. The 35°C temperature and the presence of ethanol in SSF reactions are not optimal conditions for cellulase. Immobilization onto solid supports can stabilize the enzyme and promote activity at non-optimum reaction conditions. Mock SSF reactions that did not contain yeast were used to measure saccharification products and identify the mechanism for the improved ethanol yield using immobilized cellulase. Cellulase adsorbed to 40 nm silica nanoparticles produced 1.6 times (P = 0.01) more glucose than cellulase in solution in 96 h at pH 4.8 and 35°C. There was no significant accumulation (<250 μg) of soluble cellooligomers in either the solution or immobilized enzyme reactions. This suggests that the mechanism for the immobilized enzyme's improved glucose yield compared to solution enzyme is the increased conversion of insoluble cellulose hydrolysis products to soluble cellooligomers at 35°C and in the presence of ethanol. The results show that silica-immobilized cellulase can be used to produce increased ethanol yields in the conversion of lignocellulosic materials by SSF.     

Characterization of complementary chromatic adaptation in Gloeotrichia UTEX 583 and identification of a transposon-like insertion in the cpeBA operon

Many cyanobacteria are able to alter the pigment composition of the phycobilisome in a process called complementary chromatic adaptation (CCA). The regulatory mechanisms of CCA have been identified in Fremyella diplosiphon, which regulates both phycoerythrin and phycocyanin levels, and Nostoc punctiforme, which regulates only phycoerythrin production. Recent studies show that these species use different regulatory proteins for CCA. We chose to study the CCA response of Gloeotrichia UTEX 583 in an effort to expand our knowledge about CCA and its regulation. We found that Gloeotrichia 583 has a CCA pigment response more similar to that of N. punctiforme rather than F. diplosiphon and exhibits none of the CCA-regulated morphological responses seen in F. diplosiphon. Preliminary experiments suggest that Gloeotrichia 583 contains a homolog to the CCA photoreceptor from N. punctiforme but not the CCA photoreceptor from F. diplosiphon. Additionally, two spontaneous mutants lacking phycoerythrin production were identified. Analysis has shown that these mutants contain a transposon-like insertion in the cpeA gene, which encodes the α subunit of phycoerythrin. These results suggest that CCA in Gloeotrichia UTEX 583 is more similar to that of N. punctiforme than it is to F. diplosiphon, a closely related species.