Regulation of aldoxime dehydratase activity by redox-dependent change in the coordination structure of the aldoxime-heme complex

Phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1 (OxdB) catalyzes the dehydration of Z-phenylacetaldoxime (PAOx) to produce phenylacetonitrile. OxdB contains a protoheme that works as the active center of the dehydration reaction. The enzymatic activity of ferrous OxdB was 1150-fold higher than that of ferric OxdB, indicating that the ferrous heme was the active state in OxdB catalysis. Although ferric OxdB was inactive, the substrate was bound to the ferric heme iron. Electron paramagnetic resonance spectroscopy revealed that the oxygen atom of PAOx was bound to the ferric heme, whereas PAOx was bound to the ferrous heme in OxdB via the nitrogen atom of PAOx. These results show a novel mechanism by which the activity of a heme enzyme is regulated; that is, the oxidation state of the heme controls the coordination structure of a substrate-heme complex, which regulates enzymatic activity. Rapid scanning spectroscopy using stopped-flow apparatus revealed that a reaction intermediate (the PAOx-ferrous OxdB complex) showed Soret, alpha, and beta bands at 415, 555, and 524 nM, respectively. The formation of this intermediate complex was very fast, finishing within the dead time of the stopped-flow mixer (approximately 3 ms). Site-directed mutagenesis revealed that His-306 was the catalytic residue responsible for assisting the elimination of the hydrogen atom of PAOx. The pH dependence of OxdB activity suggested that another amino acid residue that assists the elimination of the OH group of PAOx would work as a catalytic residue along with His-306.     

All four putative selectivity filter glycine residues in KtrB are essential for high affinity and selective K+ uptake by the KtrAB system from Vibrio alginolyticus

The subunit KtrB of bacterial Na+-dependent K+-translocating KtrAB systems belongs to a superfamily of K+ transporters. These proteins contain four repeated domains, each composed of two transmembrane helices connected by a putative pore loop (p-loop). The four p-loops harbor a conserved glycine residue at a position equivalent to a glycine selectivity filter residue in K+ channels. We investigated whether these glycines also form a selectivity filter in KtrB. The single residues Gly70, Gly185, Gly290, and Gly402 from p-loops P(A) to P(D) of Vibrio alginolyticus KtrB were replaced with alanine, serine, or aspartate. The three alanine variants KtrB(A70), KtrB(A185), and KtrB(A290) maintained a substantial activity in KtrAB-mediated K+ uptake in Escherichia coli. This activity was associated with a decrease in the affinity for K+ by 2 orders of magnitude, with little effect on Vmax. Minor activities were also observed for three other variants: KtrB(A402), KtrB(S70), and KtrB(D185). With all of these variants, the property of Na+ dependence of K+ transport was preserved. Only the four serine variants mediated Na+ uptake, and these variants differed considerably in their K+/Na+ selectivity. Experiments on cloned ktrB in the pBAD18 vector showed that V. alginolyticus KtrB alone was still active in E. coli. It mediated Na+-independent, slow, high affinity, and mutation-specific K+ uptake as well as K+-independent Na+ uptake. These data demonstrate that KtrB contains a selectivity filter for K+ ions and that all four conserved p-loop glycine residues are part of this filter. They also indicate that the role of KtrA lies in conferring velocity and ion coupling to the Ktr complex.     

The pgr1 mutation in the Rieske subunit of the cytochrome b6f complex does not affect PGR5-dependent cyclic electron transport around photosystem I

Although photosystem I (PSI) cyclic electron transport is essential for plants, our knowledge of the route taken by electrons is very limited. To assess whether ferredoxin (Fd) donates electrons directly to plastoquinone (PQ) or via a Q-cycle in the cytochrome (cyt) b(6)f complex in PSI cyclic electron transport, we characterized the activity of PSI cyclic electron transport in an Arabidopsis mutant, pgr1 (proton gradient regulation). In pgr1, Q-cycle activity was hypersensitive to acidification of the thylakoid lumen because of an amino acid alteration in the Rieske subunit of the cyt b(6)f complex, resulting in a conditional defect in Q-cycle activity. In vitro assays using ruptured chloroplasts did not show any difference in the activity of PGR5-dependent PQ reduction by Fd, which functions in PSI cyclic electron transport in vivo. In contrast to the pgr5 defect, the pgr1 defect did not show any synergistic effect on the quantum yield of photosystem II in crr2-2, a mutant in which NDH (NAD(P)H dehydrogenase) activity was impaired. Furthermore, the simultaneous determination of the quantum yields of both photosystems indicated that the ratio of linear and PSI cyclic electron transport was not significantly affected in pgr1. All the results indicated that the pgr1 mutation did not affect PGR5-dependent PQ reduction by Fd. The phenotypic differences between pgr1 and pgr5 indicate that maintenance of the proper balance of linear and PSI cyclic electron transport is essential for preventing over-reduction of the stroma.     

Glutathionylation of two cysteine residues in paired domain regulates DNA binding activity of Pax-8

We reported that the first two cysteine residues out of three present in paired domain (PD), a DNA-binding domain, are responsible for redox regulation of Pax-8 DNA binding activity. We show that glutathionylation of these cysteines has a regulatory role in PD binding. Wild-type PD and its mutants with substitution of cysteine to serine were synthesized and named CCC, CSS, SCS, SSC, and SSS according to the positions of substituted cysteines. They were incubated in a buffer containing various ratios of GSH/GSSG and subjected to gel shift assay. Binding of CCC, CSS, and SCS was impaired with decreasing GSH/GSSG ratio, whereas that of SSC and SSS was not affected. Because [3H]glutathione was incorporated into CCC, CSS, and SCS, but not into SSC and SSS, the binding impairment was ascribed to glutathionylation of the redox-reactive cysteines. This oxidative inactivation of PD binding was reversed by a reductant dithiothreitol and by redox factor (Ref)-1 in vitro. To explore the glutathionylation in cells, Chinese hamster ovary cells overexpressing CSS and SCS were labeled with [35S]cysteine in the presence of cycloheximide. Immunoprecipitation with an antibody against PD revealed that treatment of the cells with an oxidant diamide induced the 35S incorporation into both mutants, suggesting the PD glutathionylation in cells. Since the two cysteine residues in PD are conserved in all Pax members, this novel posttranslational modification of PD would provide a new insight into molecular basis for modulation of Pax function.     

G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.     

Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation

An optimization method for repeated fed-batch fermentation was established with the aim of improving the recombinant human serum albumin (rHSA) production in Pichia pastoris. A simulation model for fed-batch fermentation was formulated and the optimal methanol-feeding policy calculated by dynamic programming method using five different methanol-feeding periods. The necessary state variables were collected from the calculated results and used for further optimization of repeated fed-batch fermentation. The optimal operation policy was investigated using the pre-collected state variables by estimating the overall profit per total methanol-feeding time. The calculated results indicated that the initial cell mass from the 2nd fed-batch fermentation on should be set at 35 or 40 g and methanol-feeding time at 264 h. In repeated fed-batch fermentation using the optimal operation policy, actual culture volume was in good agreement with the values simulated by model equations, but some discrepancy was observed in rHSA production. Minimum experiments were therefore carried out to re-evaluate rHSA production levels, which were then applied in re-calculations to determine the optimal operation policy. The optimal policy for repeated fed-batch fermentation established in the present study (i.e., 4-times-repeated fed-batch fermentation) achieved a 47% increase in annual rHSA production. Optimization of the culture period also brought about a 28% increase in annual rHSA production even in simple (not repeated) fed-batch fermentation.     

Inhibition by adiponectin of IL-8 production by human macrophages upon coculturing with late apoptotic cells

Adiponectin, an adipocyte-derived hormone, reportedly suppresses the production of TNF-alpha and IL-6 by LPS-stimulated human or porcine macrophages, and the phagocytosis of microbeads by human macrophages. In this study, we used a high molecular weight form of adiponectin purified from human plasma to examine its effects on the phagocytosis of late apoptotic cells by human macrophages and the subsequent IL-8 production. Adiponectin suppressed both the phagocytosis of apoptotic cells and the IL-8 production. In contrast, adiponectin augmented both the phagocytosis of apoptotic cells and the IL-8 production in the presence of LPS. These results suggest that adiponectin is not an anti-inflammatory hormone but rather a dual modulator of innate responses.     

Smad3 mediates TGF-beta1 induction of VEGF production in lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is a key factor in a variety of physiological and pathological processes. Vascular endothelial growth factor (VEGF) is a key angiogenic factor, and vascular change is one of the features of airway remodeling. We examined the effect of TGF-beta1 on VEGF production by fibroblasts from mice lacking expression of Smad2 or Smad3 as well as human lung fibroblasts treated with or without Smad2 or Smad3 siRNA. TGF-beta1 stimulated VEGF production by fibroblasts from Smad2 deficient animals and wildtype animals. In contrast, TGF-beta1 did not affect VEGF production by fibroblasts from Samd3 deficient mice. Similarly, TGF-beta1 failed to stimulate VEGF production by HFL-1 cells treated with Samd3 siRNA but significantly increased VEGF production by the cells treated with Smad2 siRNA. These result suggest that TGF-beta1 stimulation of VEGF production by fibroblasts is regulated by Smad3 but not by Smad2 signaling.     

Nateglinide uptake by a ceftibuten transporter in the rat kidney brush-border membrane

Nateglinide, a novel oral hypoglycemic agent, possesses a carbonyl group and a peptide-type bond in its structure. We previously reported that nateglinide transport occurs via a single system that may be identical to the ceftibuten/H(+) cotransport system by the rat small intestine. We speculated that the absorption system present on the intestinal epithelium may be similar to that found on the renal tubular epithelium. The aim of this study was to characterize the transporters on the apical side of the kidney that may contribute to the reabsorption of ceftibuten and nateglinide. The uptake of nateglinide by rat renal brush-border membranes is associated with an H(+)-coupled transport system. Ceftibuten competitively inhibited H(+)-dependent nateglinide uptake. In contrast, Gly-Sar, cephradine and cephalexin had no effect on nateglinide uptake. Nateglinide competitively inhibited H(+)-driven transporter-mediated ceftibuten uptake. We conclude that nateglinide transport occurs via a single system that is H(+)-dependent and may be identical to the ceftibuten/H(+) cotransport system.