Project description:Polyunsaturated fatty acids are sources of diverse natural, and chemically designed products. The enzyme lipoxygenase selectively oxidizes fatty acid acyl chains using controlled free radical chemistry; the products are regio- and stereo-chemically unique hydroperoxides. A conserved structural fold of ≈600 amino acids harbors a long and narrow substrate channel and a well-shielded catalytic iron. Oxygen, a co-substrate, is blocked from the active site until a hydrogen atom is abstracted from substrate bis-allylic carbon, in a non-heme iron redox cycle. EPR spectroscopy of ferric intermediates in lipoxygenase catalysis reveals changes in the metal coordination and leads to a proposal on the nature of the reactive intermediate. Remarkably, free radicals are so well controlled in lipoxygenase chemistry that spin label technology can be applied as well. The current level of understanding of steps in lipoxygenase catalysis, from the EPR perspective, will be reviewed.
Project description:Escherichia coli possesses >65 small proteins of <50 amino acids, many of which are uncharacterized. We have identified a new small protein, MntS, involved in manganese homeostasis. Manganese is a critical micronutrient, serving as an enzyme cofactor and protecting against oxidative stress. Yet manganese is toxic in excess and little is known about its function in cells. Bacteria carefully control intracellular manganese levels using the transcription regulator MntR. Before this work, mntH, which encodes a manganese importer, was the only gene known to respond to manganese via MntR repression in E. coli K12. We demonstrated that mntS is another member of the MntR manganese regulon. We also identified yebN, which encodes a putative manganese efflux pump, as the first gene positively regulated by MntR in Enterobacteria. Since MntS is expressed when manganese levels are low, causes manganese sensitivity when overexpressed, and binds manganese, we propose that MntS may be a manganese chaperone. This study reveals new factors involved in manganese regulation and metabolism and expands our knowledge of how small proteins function. Two E. coli strains, MG1655 (wild type) and GSO458 (Delta-mntR) were grown to OD600 ~ 0.5 in M9 glucose media at 37 M-BM-:C and treated with 10 microM MnCl2. In the first experiment, this incubation with 10 microM MnCl2 was for 60 min and in the second experiment, it was for 10 min. RNA was extracted using the hot phenol method and cDNA prepared and hybridized according the manufacturer's instructions (Affymetrix).
Project description:Proton reduction and H2 oxidation are key elementary reactions for solar fuel production. Hydrogenases interconvert H+ and H2 with remarkable efficiency and have therefore received much attention in this context. For [FeFe]-hydrogenases, catalysis occurs at a unique cofactor called the H-cluster. In this article, we discuss ways in which EPR spectroscopy has elucidated aspects of the bioassembly of the H-cluster, with a focus on four case studies: EPR spectroscopic identification of a radical en route to the CO and CN- ligands of the H-cluster, tracing 57Fe from the maturase HydG into the H-cluster, characterization of the auxiliary Fe-S cluster in HydG, and isotopic labeling of the CN- ligands of HydA for electronic structure studies of its Hox state. Advances in cell-free maturation protocols have enabled several of these mechanistic studies, and understanding H-cluster maturation may in turn provide insights leading to improvements in hydrogenase production for biotechnological applications.
Project description:SLC39A14 (also known as ZIP14), a member of the SLC39A transmembrane metal transporter family, has been reported to mediate the cellular uptake of iron and zinc. Recently, however, mutations in the SLC39A14 gene have been linked to manganese (Mn) accumulation in the brain and childhood-onset parkinsonism dystonia. It has therefore been suggested that SLC39A14 deficiency impairs hepatic Mn uptake and biliary excretion, resulting in the accumulation of Mn in the circulation and brain. To test this hypothesis, we generated and characterized global Slc39a14-knockout (Slc39a14-/- ) mice and hepatocyte-specific Slc39a14-knockout (Slc39a14fl/fl;Alb-Cre+ ) mice. Slc39a14-/- mice develop markedly increased Mn concentrations in the brain and several extrahepatic tissues, as well as motor deficits that can be rescued by treatment with the metal chelator Na2CaEDTA. In contrast, Slc39a14fl/fl;Alb-Cre+ mice do not accumulate Mn in the brain or other extrahepatic tissues and do not develop motor deficits, indicating that the loss of Slc39a14 expression selectively in hepatocytes is not sufficient to cause Mn accumulation. Interestingly, Slc39a14fl/fl;Alb-Cre+ mice fed a high Mn diet have increased Mn levels in the serum, brain and pancreas, but not in the liver. Taken together, our results indicate that Slc39a14-/- mice develop brain Mn accumulation and motor deficits that cannot be explained by a loss of Slc39a14 expression in hepatocytes. These findings provide insight into the physiological role that SLC39A14 has in maintaining Mn homeostasis. Our tissue-specific Slc39a14-knockout mouse model can serve as a valuable tool for further dissecting the organ-specific role of SLC39A14 in regulating the body's susceptibility to Mn toxicity.
Project description:Escherichia coli possesses >65 small proteins of <50 amino acids, many of which are uncharacterized. We have identified a new small protein, MntS, involved in manganese homeostasis. Manganese is a critical micronutrient, serving as an enzyme cofactor and protecting against oxidative stress. Yet manganese is toxic in excess and little is known about its function in cells. Bacteria carefully control intracellular manganese levels using the transcription regulator MntR. Before this work, mntH, which encodes a manganese importer, was the only gene known to respond to manganese via MntR repression in E. coli K12. We demonstrated that mntS is another member of the MntR manganese regulon. We also identified yebN, which encodes a putative manganese efflux pump, as the first gene positively regulated by MntR in Enterobacteria. Since MntS is expressed when manganese levels are low, causes manganese sensitivity when overexpressed, and binds manganese, we propose that MntS may be a manganese chaperone. This study reveals new factors involved in manganese regulation and metabolism and expands our knowledge of how small proteins function.
Project description:We have recently developed a range of synthetic retinoid analogues which include the compounds EC23 and EC19. They are stable on exposure to light and are predicted to be resistant to the normal metabolic processes involved in the inactivation of retinoids in vivo. Based on the position of the terminal carboxylic acid groups in the compounds we suggest that EC23 is a structural analogue of all-trans retinoic acid (ATRA), and EC19 is an analogue of 13-cis retinoic acid. Their effects on the differentiation of pluripotent stem cells has been previously described in vitro and are consistent with this hypothesis. We present herein the first description of the effects of these molecules in vivo. Retinoids were applied to the anterior limb buds of chicken embryos in ovo via ion-exchange beads. We found that retinoid EC23 produces effects on the wing digits similar to ATRA, but does so at two orders of magnitude lower concentration. When larger quantities of EC23 are applied, a novel phenotype is obtained involving production of multiple digit 1s on the anterior limb. This corresponds to differential effects of ATRA and EC23 on sonic hedgehog (shh) expression in the developing limb bud. With EC23 application we also find digit 1 phenotypes similar to thumb duplications described in the clinical literature. EC23 and ATRA are shown to have effects on the entire proximal-distal axis of the limb, including hitherto undescribed effects on the scapula. This includes suppression of expression of the scapula marker Pax1. EC23 also produces effects similar to those of ATRA on the developing face, producing reductions of the upper beak at concentrations two orders of magnitude lower than ATRA. In contrast, EC19, which is structurally very similar to EC23, has novel, less severe effects on the face and rarely alters limb development. EC19 and ATRA are effective at similar concentrations. These results further demonstrate the ability of retinoids to influence embryonic development. Moreover, EC23 represents a useful new tool to investigate developmental processes and probe the mechanisms underlying congenital abnormalities in vertebrates including man.
Project description:Primordial germ cells (PGCs) give rise to the germline stem cells (GSCs) in the adult Drosophila gonads. Both PGCs and GSCs need to be tightly regulated to safeguard the survival of the entire species. During larval development, a non-cell autonomous homeostatic mechanism is in place to maintain PGC number in the gonads. Whether such germline homeostasis occurs during early embryogenesis before PGCs reach the gonads remains unclear. We have previously shown that the maternally deposited sisRNA sisR-2 can influence GSC number in the female progeny. Here we uncover the presence of a homeostatic mechanism regulating PGCs during embryogenesis. sisR-2 represses PGC number by promoting PGC death. Surprisingly, increasing maternal sisR-2 leads to an increase in PGC death, but no drop in PGC number was observed. This is due to ectopic division of PGCs via the de-repression of Cyclin B, which is governed by a genetic pathway involving sisR-2, bantam and brat. We propose a cell autonomous model whereby germline homeostasis is achieved by preserving PGC number during embryogenesis.
Project description:The P-cluster of the nitrogenase MoFe protein is a [ Fe8 S7] cluster that mediates efficient transfer of electrons to the active site for substrate reduction. Arguably the most complex homometallic FeS cluster found in nature, the biosynthetic mechanism of the P-cluster is of considerable theoretical and synthetic interest to chemists and biochemists alike. Previous studies have revealed a biphasic assembly mechanism of the two P-clusters in the MoFe protein upon incubation with Fe protein and ATP, in which the first P-cluster is formed through fast fusion of a pair of [ Fe4 S4]+ clusters within 5 min and the second P-cluster is formed through slow fusion of the second pair of [ Fe4 S4]+ clusters in a period of 2 h. Here we report a VTVH MCD and EPR spectroscopic study of the biosynthesis of the slow-forming, second P-cluster within the MoFe protein. Our results show that the first major step in the formation of the second P-cluster is the conversion of one of the precursor [ Fe4 S4]+ clusters into the integer spin cluster [ Fe4 S3-4]α, a process aided by the assembly protein NifZ, whereas the second major biosynthetic step appears to be the formation of a diamagnetic cluster with a possible structure of [ Fe8 S7-8]β, which is eventually converted into the P-cluster.
Project description:Adjusting the RedOx activity of polyoxometalate catalysts is a key challenge for the catalysis of selective oxidation reactions. For this purpose, the possibility of influencing the RedOx potential by the introduction of an additional RedOx-active element was investigated. Thereby, Keggin-type polyoxometalates (POMs) with up to three different elements in the metal framework were created. An advanced and reproducible synthetic procedure to incorporate MnII and additionally VV into Keggin-type heteropolyacids alongside comprehensive characterization of the new molecules is presented. The success of our syntheses was confirmed by vibrational spectroscopy (IR and Raman) and elemental analysis. Furthermore, the new compounds were analyzed by NMR spectroscopy to investigate the characteristics of the POMs in solution. The structures of successfully crystalized compounds were determined by single-crystal X-ray diffraction. Moreover, all synthesized compounds were characterized using UV/Vis spectroscopy and electrochemical analysis to get further insights into the electronic transfer processes and redox potentials.
Project description:Cellular levels of the essential micronutrient manganese (Mn) need to be carefully balanced within narrow borders. In cyanobacteria, a sufficient Mn supply is critical for ensuring the function of the oxygen-evolving complex as the central part of the photosynthetic machinery. However, Mn accumulation is fatal for the cells. The reason for the observed cytotoxicity is unclear. To understand the causality behind Mn toxicity in cyanobacteria, we investigated the impact of excess Mn on physiology and global gene expression in the model organism Synechocystis sp. PCC 6803. We compared the response of the WT and the knock-out mutant in the Mn exporter (Mnx), ∆mnx, which is disabled in the export of surplus Mn and thus functions as a model for toxic Mn overaccumulation. While growth and pigment accumulation in ∆mnx were severely impaired 24 h after the addition of tenfold Mn, the WT was not affected and thus mounted an adequate transcriptional response. RNA-seq data analysis revealed that the Mn stress transcriptomes partly resembled an iron limitation transcriptome. However, the expression of iron limitation signature genes isiABDC was not affected by the Mn treatment, indicating that Mn excess is not accompanied by iron limitation in Synechocystis. We suggest that the ferric uptake regulator, Fur, gets partially mismetallated under Mn excess conditions and thus interferes with an iron-dependent transcriptional response. To encounter mismetallation and other Mn-dependent problems on a protein level, the cells invest in transcripts of ribosomes, proteases and chaperones. In the case of the ∆mnx mutant, the consequences of the disability to export excess Mn from the cytosol manifest in additionally impaired energy metabolism and oxidative stress transcriptomes with a fatal outcome. This study emphasizes the central importance of Mn homeostasis and the transporter Mnx's role in restoring and holding it.