Amino acid residues in the Ler protein critical for derepression of the <Emphasis Type="Italic">LEE5</Emphasis> promoter in enteropathogenic <Emphasis Type="Italic">E. coli</Emphasis>

Enteropathogenic E. coli causes attaching and effacing (A/E) intestinal lesions. The genes involved in the formation of A/E lesions are encoded within a chromosomal island comprising of five major operons, LEE1-5. The global regulator H-NS represses the expression of these operons. Ler, a H-NS homologue, counteracts the H-NS–mediated repression. Using a novel genetic approach, we identified the amino acid residues in Ler that are involved in the interaction with H-NS: I20 and L23 in the C-terminal portion of α-helix 3, and I42 in the following unstructured linker region.     

Production of 3,6-anhydro-D-galactose from κ-carrageenan using acid catalysts

This study describes acid-catalyzed production of 3,6-anhydro-D-galactose (D-AnG) from κ-carrageenan, a sulfated polysaccharide with an alternating backbone consisting of D-AnG and D-galactose (D-Gal). We analyzed four hydrolysis products (D-AnG, 5-hydroxymethylfurfural (HMF), levulinic acid (LA), and D-Gal) and reducing sugar contents during acid hydrolysis. Acid screening was carried out using seven acid catalysts which have different acidity. The catalysts showing high D-AnG production and high selectivity were chosen for subsequent experiments. We selected four acid catalysts (HCOOH, CH₃COOH, HNO₃, and HCl), and studied the effects of catalyst acidity, hydrolysis temperature T, and reaction time t on the production of D-AnG and other hydrolysis products. The optimal condition for maximum production of D-AnG by κ-carrageenan hydrolysis was T = 100°C and t = 30 min using 0.2 M HCl. Under this condition, 2.81 g/L D-AnG (33.5% of theoretical maximum) could be obtained from 2% (w/v) κ-carrageenan. In general, the maximum values of D-AnG, D-Gal, and the sum of two by-products (HMF and LA) increased with the acidity of catalysts. However, HNO3 was an exception in that the maximum production levels of HMF and LA were unusually low compared with other acid catalysts. D-AnG was successfully purified from acid hydrolysates using silica gel chromatography and the product was nearly 100% pure. This effective D-AnG production could facilitate future studies on the conversion of D-AnG to biofuels and biochemicals.     

Effect of culture conditions on the whole cell activity of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing periplasmic organophosphorus hydrolase and cytosolic GroEL/ES chaperone

Specific whole cell activity strongly affects sensitivity and detection limit of whole cell-based biosensors. Previously, we developed recombinant Escherichia coli coexpressing periplasmic organophosphorus hydrolase (OPH) and cytosolic chaperone GroEL-GroES (GroEL/ES). In present work, we investigated the effect of culture conditions on whole cell OPH activity. Especially, the whole cell OPH activity was significantly affected by the concentration of tetracycline that is an inducer for chaperone GroEL/ES. When cultured at 20°C for 31 h in M9 medium containing 1 mM IPTG, 50 ng/mL tetracycline, and 500 µM CoCl₂, the recombinant E. coli exhibited a specific whole cell OPH activity (U/OD₆₀₀) of ~0.55, which is 2.6-fold higher than that of recombinant E. coli cultured as previously described conditions. In addition, recombinant cells showed adequate storage stability for 1 week with 100% of original response. Finally, the improved activity and adequate stability in the whole cell biocatalyst will contribute to sensitivity, detection time, and stability of a whole cell-based biosensor for the detection of toxic organophosphates.     

Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.     

Discovery of novel drug targets and their functions using phenotypic screening of natural products

Natural products are valuable resources that provide a variety of bioactive compounds and natural pharmacophores in modern drug discovery. Discovery of biologically active natural products and unraveling their target proteins to understand their mode of action have always been critical hurdles for their development into clinical drugs. For effective discovery and development of bioactive natural products into novel therapeutic drugs, comprehensive screening and identification of target proteins are indispensable. In this review, a systematic approach to understanding the mode of action of natural products isolated using phenotypic screening involving chemical proteomics-based target identification is introduced. This review highlights three natural products recently discovered via phenotypic screening, namely glucopiericidin A, ecumicin, and terpestacin, as representative case studies to revisit the pivotal role of natural products as powerful tools in discovering the novel functions and druggability of targets in biological systems and pathological diseases of interest.