Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca(2+)/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg(1400), and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.     

Structural elements involved in electron-coupled proton transfer in cytochrome c oxidase

Haem-copper oxidases are the last components of the respiratory chains in aerobic organisms. These membrane-bound enzymes energetically couple the electron transfer (eT) reactions associated with reduction of dioxygen to water, to proton pumping across the membrane. Even though the mechanism of proton pumping at the molecular level still remains to be uncovered, recent progress has presented us with the structural features of the pumping machinery and detailed information about the eT and proton-transfer reactions associated with the pumping process.     

Structural elements contribute to the calcium/calmodulin dependence on enzyme activation in human endothelial nitric-oxide synthase

Two regions, located at residues 594-606/614-645 and residues 1165-1178, are present in the reductase domain of human endothelial nitric-oxide synthase (eNOS) but absent in its counterpart, inducible nitric-oxide synthase (iNOS). We previously demonstrated that removing residues 594-606/614-645 resulted in an enzyme (Delta45) containing an intrinsic calmodulin (CaM) purified from an Sf9/baculovirus expression system (Chen, P.-F., and Wu, K.K. (2000) J. Biol. Chem. 275, 13155-13163). Here we have further elucidated the differential requirement of Ca2+/CaM for enzyme activation between eNOS and iNOS by either deletion of residues 1165-1178 (Delta14) or combined deletions of residues 594-606/614-645 and 1165-1178 (Delta45/ Delta14) from eNOS to mimic iNOS. We measured the catalytic rates using purified proteins completely free of CaM. Steady-state analysis indicated that the Delta45 supported NO synthesis in the absence of CaM at 60% of the rate in its presence, consistent with our prior result that CaM-bound Delta45 retained 60% of its activity in the presence of 10 mm EGTA. Mutant Delta14 displayed a 1.5-fold reduction of EC50 for Ca2+/CaM-dependence in l-citrulline formation, and a 2-4-fold increase in the rates of NO synthesis, NADPH oxidation, and cytochrome c reduction relative to the wild type. The basal rates of double mutant Delta45/Delta14 in NO production, NADPH oxidation, and cytochrome c reduction were 3-fold greater than those of CaM-stimulated wild-type eNOS. Interestingly, all three activities of Delta45/ Delta14 were suppressed rather than enhanced by Ca2+/CaM, indicating a complete Ca2+/CaM independence for those reactions. The results suggest that the Ca2+/CaM-dependent catalytic activity of eNOS appears to be conferred mainly by these two structural elements, and the interdomain electron transfer from reductase to oxygenase domain does not require Ca2+/CaM when eNOS lacks these two segments.     

Structural basis of charge transfer complex formation by riboflavin bound to 6,7-dimethyl-8-ribityllumazine synthase.

The amino acid residue tryptophan 27 of 6,7-dimethyl-8-ribityllumazine synthase of the yeast Schizosaccharomyces pombe was replaced by tyrosine. The structures of the W27Y mutant protein in complex with riboflavin, the substrate analogue 5-nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, and the product analogue 6-carboxyethyl-7-oxo-8-ribityllumazine, were determined by X-ray crystallography at resolutions of 2.7-2.8 A. Whereas the indole system of W27 forms a coplanar pi-complex with riboflavin, the corresponding phenyl ring in the W27Y mutant establishes only peripheral contact with the heterocyclic ring system of the bound riboflavin. These findings provide an explanation for the absence of the long wavelength shift in optical absorption spectra of riboflavin bound to the mutant enzyme. The structures of the mutants are important tools for the interpretation of the unusual physical properties of riboflavin in complex with lumazine synthase.     

Control of electron transfer in nitric-oxide synthases. Swapping of autoinhibitory elements among nitric-oxide synthase isoforms

To clarify the role of the autoinhibitory insert in the endothelial (eNOS) and neuronal (nNOS) nitric-oxide synthases, the insert was excised from nNOS and chimeras with its reductase domain; the eNOS and nNOS inserts were swapped and put into the normally insertless inducible (iNOS) isoform and chimeras with the iNOS reductase domain; and an RRKRK sequence in the insert suggested by earlier peptide studies to be important (Salerno, J. C., Harris, D. E., Irizarry, K., Patel, B., Morales, A. J., Smith, S. M., Martasek, P., Roman, L. J., Masters, B. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q., and Gross, S. S. (1997) J. Biol. Chem. 272, 29769-29777) was mutated. Insertless nNOS required calmodulin (CaM) for normal NOS activity, but the Ca(2+) requirement for this activity was relaxed. Furthermore, insert deletion enhanced CaM-free electron transfer within nNOS and chimeras with the nNOS reductase, emphasizing the involvement of the insert in modulating electron transfer. Swapping the nNOS and eNOS inserts gave proteins with normal NOS activities, and the nNOS insert acted normally in raising the Ca(2+) dependence when placed in eNOS. Insertion of the eNOS insert into iNOS and chimeras with the iNOS reductase domain significantly lowered NOS activity, consistent with inhibition of electron transfer by the insert. Mutation of the eNOS RRKRK to an AAAAA sequence did not alter the eNOS Ca(2+) dependence but marginally inhibited electron transfer. The salt dependence suggests that the insert modulates electron transfer within the reductase domain prior to the heme/reductase interface. The results clarify the role of the reductase insert in modulating the Ca(2+) requirement, electron transfer rate, and overall activity of nNOS and eNOS.     

Sucrose synthase localizes to cellulose synthesis sites in tracheary elements.

The synthesis of crystalline cellulose microfibrils in plants is a highly coordinated process that occurs at the interface of the cortex, plasma membrane, and cell wall. There is evidence that cellulose biogenesis is facilitated by the interaction of several proteins, but the details are just beginning to be understood. In particular, sucrose synthase, microtubules, and actin have been proposed to possibly associate with cellulose synthases (microfibril terminal complexes) in the plasma membrane. Differentiating tracheary elements of Zinnia elegans L. were used as a model system to determine the localization of sucrose synthase and actin in relation to the plasma membrane and its underlying microtubules during the deposition of patterned, cellulose-rich secondary walls. Cortical actin occurs with similar density both between and under secondary wall thickenings. In contrast, sucrose synthase is highly enriched near the plasma membrane and the microtubules under the secondary wall thickenings. Both actin and sucrose synthase lie closer to the plasma membrane than the microtubules. These results show that the preferential localization of sucrose synthase at sites of high-rate cellulose synthesis can be generalized beyond cotton fibers, and they establish a spatial context for further work on a multi-protein complex that may facilitate secondary wall cellulose synthesis.     

Channeling of ammonia in glucosamine-6-phosphate synthase.

Glucosamine-6-phosphate synthase catalyses the first and rate-limiting step in hexosamine metabolism, converting fructose 6-phosphate into glucosamine 6-phosphate in the presence of glutamine. The crystal structure of the Escherichia coli enzyme reveals the domain organisation of the homodimeric molecule. The 18 A hydrophobic channel sequestered from the solvent connects the glutaminase and isomerase active sites, and provides a means of ammonia transfer from glutamine to sugar phosphate. The C-terminal decapeptide sandwiched between the two domains plays a central role in the transfer. Based on the structure, a mechanism of enzyme action and self-regulation is proposed. It involves large domain movements triggered by substrate binding that lead to the formation of the channel.     

Effect of ammonia on sucrose synthase in pea roots

The effects of ammonium on activity of sucrose synthase (SS) in the roots of pea (Pisum sativum L.) plants were studied. On the medium containing 14.2 mM (NH₄)₂SO₄, SS activity increased by 20–200% for 10–20 days of plant growth as compared with the roots of plants growing without nitrogen. Illuminance affected the degree of effects. Under natural illumination, ammonium affected SS activity not only in sunny days (up to 25 klx) but also in cloudy days (3–6 klx) but to a lower degree. Under stable low light (2.5 klx), ammonium did not affect SS activity. In the in vitro experiments, at (NH₄)₂SO₄ concentrations from 0 to 1 mM, SS activity was suppressed (up to 10%), whereas 1–37.5 mM (NH₄)₂SO₄, it was increased (up to 50%).     

Structural elements common to mitosis and apoptosis.

Both mitotic and apoptotic cells display hypercondensation of the chromatin and loss of the nuclear envelope (Lazebnik et al., 1993). Herein, we describe a third similarity between the two processes. We have observed, initially in apoptotic cells of the PC-12 lineage clusters of 40-60 (approximately 50) nm vesicles adjoined by a minor contingent of tubule vesicular elements of 100-200 nm which are indistinguishable from their vesicular counterparts in mitotic PC-12 cells. The clusters of approximately 50 nm vesicles were subsequently observed in all studied rat tissue cells in apoptosis (plasma cells and macrophages, secretory epithelial cells from pancreatic acini, ventral lobe of prostate and mammary gland). Clusters of approximately 50 nm vesicles comparable to those of the PC-12 cells were found in HeLa cells treated with human alfa TNF, in WEHI-3 cells exposed to VM 26 (a teneposide) (Sesso et al., 1997) and in HL-60 cells treated with thapsigargin. PC-12 and HeLa cells affixed to coverslips were double labelled and examined with the fluorescence microscope to reveal simultaneously the disposition of the chromatin with Hoechst stain and the distribution of the fluorescence of Golgi or of Golgi-associated proteins. A common pattern of fluorescence was observed in a minor proportion of apoptotic cells using three different antibodies used. The label frequently appeared as finely dispersed granules in the cytoplasm. In some apoptotic cells, relatively coarse granules were observed. This pattern of label distribution is compatible with the disposition of vesicular clusters we have encountered in apoptotic PC-12 cells sectioned serially or semi serially. In such sections of both mitotic and apoptotic PC-12 cells, we noticed that the conglomerates of 50 nm vesicles were frequently associated with cisternae of the rough ER. Vesicles of similar size were also noted pinching off from the extremities of Golgi cisternae reduced in size. These cisternae diminish in length and width when they are in the process of disassembling at the very beginning of mitosis and in apoptosis.     

Responses of wetland plants to ammonia and water level

Constructed wetland systems receiving animal wastewater may enhance water quality when designed, operated, and maintained properly. In the case of wetlands designed to treat animal waste, system effectiveness may be limited by high ammonia concentrations and inundation, conditions that can adversely affect macrophytic vegetation. We conducted a 4-month greenhouse experiment to assess the impact of ammonia concentration and water level on plants commonly used in constructed wetlands for treating animal waste. We examined the effects of ammonia concentration (0, 50, 100, 200 and 400 mg/l) on the growth and biomass production of Juncus effusus, Sagittaria latifolia, Schoenoplectus tabernaemontani, Typha angustifolia, and Typha latifolia. We also explored interactions between ammonia concentration (0, 50, 100, 200 and 400 mg/l) and water level (flooded and nonflooded conditions) for S. tabernaemontani and T. latifolia. We found that ammonia levels in excess of 200 mg/l inhibited growth for J. effusus, S. latifolia, and T. latifolia after a period of weeks, and levels in excess of 100 mg/l similarly inhibited growth for S. tabernaemontani. Ammonia levels in the range studied had an ambiguous effect on T. angustifolia. Affected species demonstrated similar fertilization/inhibition responses to increased ammonia, but important differences were noted between species. Flooded conditions of 10 cm did not significantly increase ammonia toxicity to S. tabernaemontani or T. latifolia. Our results emphasize the need for careful consideration of the species used in treatment wetlands, and suggest that management of ammonia concentration may enhance plant growth and system function.     

Structural basis for endothelial nitric oxide synthase binding to calmodulin

The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS.     

Substrate-induced changes in the ammonia channel for imidazole glycerol phosphate synthase

IGP synthase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the unusual nucleotide, N(1)-[(5'-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to form 5'-(5-aminoimidazole-4-carboxamide) ribonucleotide (AICAR) and imidazole glycerol phosphate (IGP). A common feature of all glutamine amidotransferases is the upregulation of glutamine hydrolysis in the presence of an acceptor substrate. A refined assay system was developed to establish that Saccharomyces cerevisae IGP synthase shows a 4900-fold stimulation of glutaminase in the presence of the substrate acceptor PRFAR. The structure and function of IGP synthase acceptor substrate binding site were probed with competitive inhibitors that are nucleotide substrate and product analogues. In addition, these analogues were also used to establish that the normal steady-state turnover cycle involves a random sequential mechanism. Upregulation of the glutaminase active site occurs when these competitive inhibitors bind in the nucleotide site over 30 A away. One of the key structural features of IGP synthase is that the transfer of ammonia from the glutaminase site occurs through the (beta/alpha)(8) core of the protein. Upon the basis of the recent substrate-occupied structure for yeast IGP synthase (1), kinetic investigations of site-directed mutants revealed that a conserved K258 residue is key to productive binding and the overall stoichiometry of the reaction. The binding of the ribulosyl phosphate portion of the substrate PRFAR appears to be transduced through reorientation of K258 resulting in a conformational switch at the base of the (beta/alpha)(8) core that enables the passage of ammonia through the core of the protein. The overall analysis also leads to further discussion of how the residues that cover the opening of the (beta/alpha)(8) in the closed state may assist the channeling of ammonia at the interface of the two functional domains in the open state.     

Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission

Imaging of fluorescence resonance energy transfer (FRET) between suitable fluorophores is increasingly being used to study cellular processes with high spatiotemporal resolution. The genetically encoded Cyan (CFP) and Yellow (YFP) variants of Green Fluorescent Protein have become the most popular donor and acceptor pair in cell biology. FRET between these fluorophores can be imaged by detecting sensitized emission. This technique, for which CFP is excited and transfer is detected as emission of YFP, is sensitive, fast, and straightforward, provided that proper corrections are made. In this study, the detection of sensitized emission between CFP and YFP by confocal microscopy is optimized. It is shown that this FRET pair is best excited at 430 nm. We identify major sources of error and variability in confocal FRET acquisition including chromatic aberrations and instability of the excitation sources. We demonstrate that a novel correction algorithm that employs online corrective measurements yields reliable estimates of FRET efficiency, and it is also shown how the effect of other error sources can be minimized.     

Promoter elements in the aflatoxin pathway polyketide synthase gene

PksA catalyzes the formation of the polyketide backbone necessary for aflatoxin biosynthesis. Based on reporter assays and sequence comparisons of the nor1-pksA intergenic region in different aflatoxin-producing Aspergillus species, cis-acting elements for the aflatoxin pathway-specific regulatory protein, AflR, and the global-acting regulatory proteins BrlA and PacC are involved in pksA promoter activity.     

Electron transfer by neuronal nitric-oxide synthase is regulated by concerted interaction of calmodulin and two intrinsic regulatory elements

The nitric-oxide synthases (NOSs) are modular, cofactor-containing enzymes, divided into a heme-containing oxygenase domain and an FMN- and FAD-containing reductase domain. The domains are connected by a calmodulin (CaM)-binding sequence, occupancy of which is required for nitric oxide (NO) production. Two additional CaM-modulated regulatory elements are present in the reductase domains of the constitutive isoforms, the autoregulatory region (AR) and the C-terminal tail region. Deletion of the AR reduces CaM stimulation of electron flow through the reductase domain from 10-fold in wild-type nNOS to 2-fold in the mutant. Deletion of the C terminus yields an enzyme with greatly enhanced reductase activity in the absence of CaM but with activity equivalent to that of wild-type enzyme in its presence. A mutant in which both the AR and C terminus were deleted completely loses CaM modulation through the reductase domain. Thus, transduction of the CaM effect through the reductase domain of nNOS is dependent on these elements. Formation of nitric oxide is, however, still stimulated by CaM in all three mutants. A CaM molecule in which the N-terminal lobe was replaced by the C-terminal lobe (CaM-CC) supported NO synthesis by the deletion mutants but not by wild-type nNOS. We propose a model in which the AR, the C-terminal tail, and CaM interact directly to regulate the conformational state of the reductase domain of nNOS.     

优化滩涂养殖水体生态结构和调节水质的研究

采用在滩涂鱼塘和虾池中接种小球藻和有益微生物的方法,探讨了调控滩涂养殖水体生态结构的浮游生物种群、数量、生物量及化学环境的可行性。结果表明,虾池和鱼塘中接种小球藻后,小球藻数量均大幅度增加,变为优势种群,分别是其对照的16.92倍、4.76倍;浮游动物生物量为4.32mg·L^-1和2.84mg·L^-1,分别比对照增加19.3%、2.5%,同样,光合细菌、酵母菌及硝化细菌显著地改变了水体藻类和浮游动物种群的组成、数量、比例及生物量等,“酵母菌＋硝化细菌”的处理,使水体氨态氮浓度下降最明显,为对照的44％,虾池生化耗氧和化学耗氧,为对照的56.5%和38.4%,增加了水体溶解氧和初级生产力。     

Maternal perinatal transfer of vitamins and trace elements to piglets

Nursing piglets are entirely dependent, for their micronutrient provisions, upon in utero, colostrum and milk transfers from the dam. An adequate maternal transfer of micronutrients is all the more important during these periods which, in fact, lasts for approximately half the life cycle (conception to slaughter) of modern pigs. The present study aimed to set up a simple approach to assess the maternal perinatal transfer of vitamins and trace elements in sows. Prenatal transfer (R-u) was estimated as limited, passive or active using the ratio between pre-colostral serum concentrations of a given micronutrient in newborn piglets and corresponding pre-farrowing values in sows. Efficiency of the postnatal transfer (R-c) was estimated from the ratio between serum concentrations of post- and pre-colostral micronutrients in piglets. Data from literature (12 studies) were used for vitamins A, D, E, C, folic acid and B₁₂, whereas vitamins B₂, B₃, B₆ and B₈ as well as Zn, Fe, Cu and Se were generated from a trial where blood sera from 20 sows, and their litter were collected during the perinatal period. In sow trial, statistical t tests were used to determine if ratios differed from 1. Prenatal transfer was active and in favour of piglets (R-u > 1, P < 0.03) for Zn and vitamins B₆ and B₈ (sow trial) as well as for vitamins C and B₁₂ (literature data). This transfer was limited (R-u < 1, P < 0.01) for vitamin B₂, Fe, Cu and Se (sow trial) and for vitamins A, E, D and folic acid (literature data) whereas it was passive for vitamin B₃ (R-u = 1, P > 0.37). After birth, the early postnatal transfer through colostrum was active towards piglets for most micronutrients but vitamins B₆ and B₈ (R-c < 1, P < 0.01). Globally, the perinatal transfer (combination of R-u and R-c) was favourable to the neonatal piglets for most micronutrients except for vitamins A and D as well as Fe, Cu and Se whereas there is apparently a barrier for prenatal transfer which is not compensated by the colostrum provision to neonatal piglets. Then, post-colostral concentrations of these micronutrients in piglets remain below prenatal levels of their dam. Neonatal strategies of micronutrient provision are known for Fe (intramuscular injection) and Se (sow milk enrichment). Further studies are needed to assess the importance of the unfavourable perinatal transfer for Cu and vitamins A and D for piglet robustness later in life.     

Material transfer from water to sediment in Florida lakes

We describe statistical relationships between chemical aspects of surficial sediments from 34 Florida lakes, and trophic state of the overlying waters. Trophic state is expressed by Carlson's TSI-Chl a. The objectives of the study are two-fold: 1) to understand processes that govern the transfer of material across the mudwater interface, and 2) to develop transfer functions for inferring historical TSI measures in chemically analyzed, ²¹⁰Pb-dated cores. Simple regression of organic matter content or nutrient (C, N, P) concentration in surface sediments vs. TSI yields nonsignificant or weak positive correlations. However, using a novel application of ²¹⁰Pb assay, net accumulation rates of the materials are estimated and show a better correlation with trophic state. Cation (Ca, Mg, Fe, K) and sulfur concentrations in surface muds are poorly related to corresponding TSI's. Net accumulation rates of these elements (Ca, Mg, Fe, K, S) are positively correlated (P < 0.01) with TSI-Chl a. Chemical data from the Florida surficial sediment survey suggest that inferred levels of past trophic state should be based on net accumulation rates of chemical constituents rather than their simple sediment concentrations.     

Photorespiratory ammonia does not inhibit photosynthesis in glutamate synthase mutants of Arabidopsis

Exposure of ferredoxin-dependent glutamate synthase (EC 1.4.7.1) mutants of Arabidopsis thaliana to photorespiratory conditions resulted in the accumulation of NH₄⁺ and the inhibition of photosynthesis. However, upon transfer from 2% O₂, 350 microliters per liter CO₂, to 21% O₂, 350 microliters per liter CO₂, net photosynthesis declined at a slower rate in methionine sulfoximine treated leaf discs relative to controls. The recovery of photosynthesis was also more rapid in MSO-treated leaf discs although ammonia levels were more than threefold higher. Photosynthesis in leaf discs treated with azaserine was inhibited more than controls when transferred to 21% O₂ and recovered less than controls when returned to 2% O₂ although NH₄⁺ levels were not significantly different. The results obtained are consistent with the view that the rapid inhibition of photosynthesis in the glutamate synthase mutants in photorespiratory conditions is not due to the accumulation of NH₄⁺ but rather to the depletion of amino donors for glyoxylate and the consequent effects of glyoxylate on the lack of return of carbon to the chloroplast.