Functional characterization and molecular modeling of methylcatechol 2,3-dioxygenase from o-xylene-degrading Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 is known to metabolize o-xylene and toluene through the intermediates 3,4-dimethylcatechol and 3- and 4-methylcatechol, respectively, which are further cleaved by a common catechol 2,3-dioxygenase. A putative gene encoding this enzyme (akbC) was amplified by PCR, cloned, and expressed in Escherichia coli. Assessment of the enzyme activity expressed in E. coli combined with sequence analysis of a mutant gene demonstrated that the akbC gene encodes the bona fide catechol 2,3-dioxygenase (AkbC) for metabolism of o-xylene and alkylbenzenes such as toluene and ethylbenzene. Analysis of the deduced amino acid sequence indicates that AkbC consists of a new catechol 2,3-dioxygenase class specific for methyl-substituted catechols. A computer-aided molecular modeling studies suggest that amino acid residues (particularly Phe177) in the beta10-beta11 loop play an essential role in characterizing the substrate specificity of AkbC.     

Differential CCR1-mediated chemotaxis signaling induced by human CC chemokine HCC-4/CCL16 in HOS cells

Human CC chemokine-4 (HCC-4)/CCL16 is a chemoattractant for monocytes and lymphocytes. Although HCC-4 binds to multiple CC chemokine receptors, the receptor-mediated signal transduction pathway induced by HCC-4 has not been characterized. Human osteogenic sarcoma cells stably expressing CCR1 were used to investigate HCC-4-mediated chemotaxis signaling events via CCR1. The chemotactic activity of HCC-4 as well as those of other CCR1-dependent chemokines including MIP-1alpha/CCL3, RANTES/CCL5, and Lkn-1/CCL15 was inhibited by the treatment of pertussis toxin, an inhibitor of Gi/Go protein, U73122, an inhibitor of phospholipase C (PLC), and rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta). These results indicate that HCC-4-induced chemotaxis signaling is mediated through Gi/Go protein, PLC, and PKCdelta. SB202190, an inhibitor of p38 mitogen activated protein kinase, only blocked the chemotactic activity of HCC-4, but not those of other CCR1-dependent chemokines. SB202190 inhibited HCC-4-induced chemotaxis in a dose-dependent manner (P < 0.01). HCC-4 induces p38 activation in both a time and dose-dependent manner. However, such p38 activation was not induced by other CCR1-dependent chemokines. To further investigate the differential effect of HCC-4, the Ca2+ mobilization was examined. HCC-4 induced no intracellular Ca2+ flux in contrast to other CCR1-dependent chemokines. These results indicate that HCC-4 transduces signals differently from other CCR1-dependent chemokines and may play different roles in the immune response.     

Identification of parathyroid hormone-regulated proteins in mouse bone marrow cells by proteomics

The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. Several studies have suggested that the activation of bone marrow stromal cells could be preceded to show the anabolic effect of PTH on bone formation, but little is known of PTH-regulated proteins in bone marrow cells. Therefore, protein profiling in the intermittent PTH-treated bone marrow cells was evaluated using proteomics. Daily treatment for 5 days consisting of subcutaneous injection of either 150 microg/kg per day of mouse PTH (1-84) or vehicle (0.9% normal saline) was performed on the ICR mouse. At the end of the treatment period, bone marrow cells were separated and used in proteomics. The expression levels of seven proteins including vimentin were decreased, but those of four proteins including calreticulin and thioredoxin domain containing 7 protein (Txnde7) were increased. Among these, the decrease of vimentin and the increase of both calreticulin Txnde7 in mRNA levels were confirmed by semi-quantitative RT-PCR. In PTH-treated mouse MC3T3-E1 osteoblast cells, mRNA expression levels were not totally consistent with the results observed in proteomics. In conclusion, the differentially expressed proteins in bone marrow cells depending on PTH could be highly linked to the differentiation of osteoprogenitor cells in the bone marrow into preosteoblast cells.     

Effect of jasmonic acid on endogenous gibberellins and abscisic acid in rice under NaCl stress

Content of endogenous abscisic acid (ABA) increased in rice plants under salt stress. Pre- or post-treatment by jasmonic acid (JA) mostly further increased ABA content. In the presence of salt stress also content of gibberellins (GAs) mostly increased more after treatment by JA. Endogenous content of bioactive GA₁ was higher in post-treatment by JA than in pre-treatment by JA.This study was supported by the Korea Science and Engineering Foundation (2000-2-20100-001-3)     

Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni

Equilibrium and kinetic analyses have been carried out to elucidate the folding mechanism of homodimeric ketosteroid isomerase (KSI) from Comamonas testosteroni. The folding of KSI was reversible since the activity as well as the fluorescence and CD spectra was almost completely recovered after refolding. The equilibrium unfolding transitions monitored by fluorescence and CD measurements were almost coincident with each other, and the transition midpoint increased with increasing protein concentration. This suggests that the KSI folding follows a simple two-state mechanism consisting of native dimer and unfolded monomer without any thermodynamically stable intermediates. Sedimentation equilibrium analysis and size-exclusion chromatography of KSI at different urea concentrations supported the two-state model without any evidence of folded monomeric intermediates. Consistent with the two-state model, (1)H-(15)N HSQC spectra obtained for KSI in the unfolding transition region could be reproduced by a simple addition of the spectra of the native and the unfolded KSI. The KSI refolding kinetics as monitored by fluorescence intensity could be described as a fast first-order process followed by a second-order and a subsequent slow first-order processes with rate constants of 60 s(-)(1), 5.4 x 10(4) M(-)(1).s(-)(1), and 0.017 s(-)(1), respectively, at 0.62 M urea, suggesting that there may be a monomeric folding intermediate. After a burst phase that accounts for >83% of the total amplitude, the negative molar ellipticity at 225 nm increased slowly in a single phase at a rate comparable to that of the bimolecular intermediate step. The kinetics of activity recovery from the denatured state were markedly dependent upon the protein concentration, implying that the monomers are not fully active. Taken together, our results demonstrate that the dimerization induces KSI to fold into the complete structure and is crucial for maintaining the tertiary structure to perform efficient catalysis.     

Effects of the hinge region of cecropin A(1-8)-magainin 2(1-12), a synthetic antimicrobial peptide, on liposomes, bacterial and tumor cells

A 20-residue hybrid peptide (CA(1-8)-MA(1-12): KWKLFKKIGIGKFLHSAKKF-NH(2)) incorporating 1-8 residues of cecropin A (CA) and 1-12 residues of magainin 2 (MA) has potent antibiotic activity without hemolytic activity. In order to investigate the effects of the flexible hinge sequence, Gly-Ile-Gly of CA(1-8)-MA(1-12) (CA-MA) on antibiotic activity, CA-MA and its three analogues, CA-MA1, CA-MA2 and CA-MA3 were synthesized. The Gly-Ile-Gly sequence of CA-MA was deleted in CA-MA1 and replaced with Pro and Gly-Pro-Gly in CA-MA2 and CA-MA3, respectively. CA-MA1 and CA-MA3 caused a significant decrease in the bactericidal rate against Escherichia coli and Bacillus subtilis and the tumoricidal activity against four different tumor cells, and the PC/PS (4:1, w/w) vesicle-aggregating and disrupting activities. However, CA-MA2 showed a similar bactericidal rate and antitumor, vesicle-aggregating and disrupting activities, as compared with CA-MA. These results suggested that the flexibility or beta-turn induced by Gly-Ile-Gly or Pro in the central part of CA-MA may be important in the electrostatic interaction of the cationic short alpha-helical region in the N-terminus with the cell membrane surface and the hydrophobic interaction of amphipathic alpha-helical region in the C-terminus with the hydrophobic acyl chains in the cell membrane. CA-MA3 exhibited lower activity in antibacterial, antitumor, and vesicle-aggregating and disrupting activities than CA-MA and CA-MA2. This result suggested that the excessive beta-turn structure by Gly-Pro-Gly in CA-MA3 seems to interrupt the ion channel/pore formation on the lipid bilayer. It was concluded that the appropriate flexibility or beta-turn structure provided by the central hinge is responsible for the effective antibiotic activity of the antimicrobial peptides with the helix-hinge-helix structure.     

Gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in Drosophila melanogaster cells

Pattern recognition receptors, non-clonal immune proteins recognizing common microbial components, are critical for non-self recognition and the subsequent induction of Rel/NF-kappaB-controlled innate immune genes. However, the molecular identities of such receptors are still obscure. Here, we present data showing that Drosophila possesses at least three cDNAs encoding members of the Gram-negative bacteria-binding protein (DGNBP) family, one of which, DGNBP-1, has been characterized. Western blot, flow cytometric, and confocal laser microscopic analyses demonstrate that DGNBP-1 exists in both a soluble and a glycosylphosphatidylinositol-anchored membrane form in culture medium supernatant and on Drosophila immunocompetent cells, respectively. DGNBP-1 has a high affinity to microbial immune elicitors such as lipopolysaccharide (LPS) and beta-1,3-glucan whereas no binding affinity is detected with peptidoglycan, beta-1,4-glucan, or chitin. Importantly, the overexpression of DGNBP-1 in Drosophila immunocompetent cells enhances LPS- and beta-1,3-glucan-induced innate immune gene (NF-kappaB-dependent antimicrobial peptide gene) expression, which can be specifically blocked by pretreatment with anti-DGNBP-1 antibody. These results suggest that DGNBP-1 functions as a pattern recognition receptor for LPS from Gram-negative bacteria and beta-1, 3-glucan from fungi and plays an important role in non-self recognition and the subsequent immune signal transmission for the induction of antimicrobial peptide genes in the Drosophila innate immune system.     

Radiolytic studies of trimethylamine dehydrogenase. Spectral deconvolution of the neutral and anionic flavin semiquinone, and determination of rate constants for electron transfer in the one-electron reduced enzyme

Trimethylamine dehydrogenase from the pseudomonad Methylophilus methylotrophus has been examined using the technique of pulse radiolysis to rapidly introduce a single reducing equivalent into the enzyme. Using enzyme that has had its iron-sulfur center rendered redox-inert by prior reaction with ferricenium hexafluorophosphate, we determined the spectral change associated with formation of both the anionic and neutral forms that were generated at high and low pH, respectively, of the unique 6-cysteinyl-FMN of the enzyme. With native enzyme, electron transfer was observed within the radiolytically generated one-electron reduced enzyme but only at low pH (6.0). The kinetics and thermodynamics of this electron transfer in one-electron reduced enzyme may be compared with that studied previously in the two-electron reduced enzyme. In contrast to previous studies with two-electron reduced enzyme in which a pK(a) of approximately 8 was determined for the flavin semiquinone, in the one-electron reduced enzyme the semiquinone was not substantially protonated even at pH 6. 0. These results indicate that reduction of the iron-sulfur center of the enzyme significantly decreases the pK(a) of the flavin semiquinone of the active site. This provides further evidence, in conjunction with the strong magnetic interaction known to exist between the centers in the two-electron reduced enzyme, that the two redox-active centers in trimethylamine dehydrogenase are in intimate contact with one another in the active site of the enzyme.