Structure and reaction geometry of geranylgeranyl diphosphate synthase from Sinapis alba

The crystal structure of the geranylgeranyl diphosphate synthase from Sinapis alba (mustard) has been solved in two crystal forms at 1.8 and 2.0 A resolutions. In one of these forms, the dimeric enzyme binds one molecule of the final product geranylgeranyl diphosphate in one subunit. The chainfold of the enzyme corresponds to that of other members of the farnesyl diphosphate synthase family. Whereas the binding modes of the two substrates dimethylallyl diphosphate and isopentenyl diphosphate at the allyl and isopentenyl sites, respectively, have been established with other members of the family, the complex structure presented reveals for the first time the binding mode of a reaction product at the isopentenyl site. The binding geometry of substrates and product in conjunction with the protein environment and the established chemistry of the reaction provide a clear picture of the reaction steps and atom displacements. Moreover, a comparison with a ligated homologous structure outlined an appreciable induced fit: helix alpha8 and its environment undergo a large conformational change when either the substrate dimethylallyl diphosphate or an analogue is bound to the allyl site; only a minor conformational change occurs when the other substrate isopentenyl diphosphate or the product is bound to the isopentenyl site.     

Ceramic methyltrioxorhenium

The metal oxide polymeric methyltrioxorhenium [(CH₃)_xReO₃]_∞ is a unique representative of a layered inherent-conducting organometallic polymer which adopts the structural motifs of classical perovskites in two dimensions (2D) in form of methyl-deficient, corner-sharing ReO₅(CH₃) octahedra. In order to improve the characteristics of polymeric methyltrioxorhenium with respect to its physical properties and potential usage as an inherent-conducting polymer we tried to optimise the synthetic routes of polymeric modifications of 1 to obtain a sintered ceramic material, denoted as ceramic MTO. Ceramic MTO formed in a solvent-free synthesis via auto-polymerisation and subsequent sintering processing displays clearly different mechanical and physical properties from polymeric MTO synthesised in aqueous solution. Ceramic MTO is shown to display activated ReC and ReO bonds relative to MTO. These electronic and structural characteristics of ceramic MTO are also reflected by a different chemical reactivity compared with its monomeric parent compound. First examples of the unprecedented reactivity of ceramic MTO in the field of amine oxidations are shown - results which warrant further exploitation.     

Aerobic benzoyl-coenzyme A (CoA) catabolic pathway in Azoarcus evansii: conversion of ring cleavage product by 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase

Benzoate, a strategic intermediate in aerobic aromatic metabolism, is metabolized in various bacteria via an unorthodox pathway. The intermediates of this pathway are coenzyme A (CoA) thioesters throughout, and ring cleavage is nonoxygenolytic. The fate of the ring cleavage product 3,4-dehydroadipyl-CoA semialdehyde was studied in the beta-proteobacterium Azoarcus evansii. Cell extracts contained a benzoate-induced, NADP(+)-specific aldehyde dehydrogenase, which oxidized this intermediate. A postulated putative long-chain aldehyde dehydrogenase gene, which might encode this new enzyme, is located on a cluster of genes encoding enzymes and a transport system required for aerobic benzoate oxidation. The gene was expressed in Escherichia coli, and the maltose-binding protein-tagged enzyme was purified and studied. It is a homodimer composed of 54 kDa (without tag) subunits and was confirmed to be the desired 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase. The reaction product was identified by nuclear magnetic resonance spectroscopy as the corresponding acid 3,4-dehydroadipyl-CoA. Hence, the intermediates of aerobic benzoyl-CoA catabolic pathway recognized so far are benzoyl-CoA; 2,3-dihydro-2,3-dihydroxybenzoyl-CoA; 3,4-dehydroadipyl-CoA semialdehyde plus formate; and 3,4-dehydroadipyl-CoA. The further metabolism is thought to lead to 3-oxoadipyl-CoA, the intermediate at which the conventional and the unorthodox pathways merge.     

Properties of succinyl-coenzyme A:L-malate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large (alphabeta)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate --> succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle.     

Saccharomyces cerevisiae strain expressing a plant fatty acid desaturase produces polyunsaturated fatty acids and is susceptible to oxidative stress induced by lipid peroxidation

Although oxygen is essential for aerobic organisms, it also forms potentially harmful reactive oxygen species. For its simplicity, easy manipulation, and cultivation conditions, yeast is used as an attractive model in oxidative stress research. However, lack of polyunsaturated fatty acids in yeast membranes makes yeast unsuitable for research in the field of lipid peroxidation. Therefore, we have constructed a yeast strain expressing a Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This yeast strain expresses the heterologous desaturase in an active form and, consequently, produces Delta9/Delta12 polyunsaturated fatty acids under inducing conditions. The functional expression of the heterologous desaturase did not affect cellular morphology or growth, indicating no general adverse effect on cellular physiology. However, the presence of polyunsaturated fatty acids changed the yeast's sensitivity to oxidative stress induced by addition of paraquat, tert-butylhydroperoxide, and hydrogen peroxide. This difference in sensitivity to the latter was followed by the formation of 4-hydroxy-2-nonenal, one of the end products of linoleic fatty acid peroxidation, which is known to play a role in cell growth control and signaling. Here we show that this yeast strain conditionally expressing the Delta12 desaturase gene provides a novel and well-defined eukaryotic model in lipid peroxidation research. Its potential to investigate the molecular basis of responses to oxidative stress, in particular the involvement of reactive aldehydes derived from fatty acid peroxidation, especially 4-hydroxy-2-nonenal, will be addressed.     

Seasonality Pattern of Biomass Accumulation in a Drifting Furcellaria Lumbricalis Community in the Waters of the West Estonian Archipelago, Baltic Sea

A free-floating, loose form of Furcellaria lumbricalis (Huds.) Lamour is rare in the Baltic Sea area. Kassari Bay, situated in the West Estonian Archipelago Sea area contains the largest known community of this kind. Here the free-floating mixed Furcellaria lumbricalis-Coccotylus truncatus (Paela) M. J. Wynne et J. N. Heine community inhabits sandy bottom, covering up to 120 km². Commercial exploitation of the community started in 1966 and has led to regular monitoring surveys for the quantification of the commercial resource. The aim of the present study was to determine the potential growth rates of the two community-forming species as well as to test different environmental factors affecting their growth. Results showed that the highest growth rates were measured in shallower depths (4 m) for both species. The seasonal growth pattern was also very similar for both species, showing the highest growth rates during the beginning of summer. Incubation of both species in another sea area with apparently similar basic environmental conditions (the northern part of the Gulf of Riga, Kõiguste Bay) resulted in significantly lower growth rates during the whole incubation period.     

Adaptive Mutations Resulting in Enhanced Polymerase Activity Contribute to High Virulence of Influenza A Virus in Mice

High virulence of influenza virus A/Puerto Rico/8/34 in mice carrying the Mx1 resistance gene was recently shown to be determined by the viral surface proteins and the viral polymerase. Here, we demonstrated high-level polymerase activity in mammalian host cells but not avian host cells and investigated which mutations in the polymerase subunits PB1, PB2, and PA are critical for increased polymerase activity and high virus virulence. Mutational analyses demonstrated that an isoleucine-to-valine change at position 504 in PB2 was the most critical and strongly enhanced the activity of the reconstituted polymerase complex. An isoleucine-to-leucine change at position 550 in PA further contributed to increased polymerase activity and high virulence, whereas all other mutations in PB1, PB2, and PA were irrelevant. To determine whether this pattern of acquired mutations represents a preferred viral strategy to gain virulence, two independent new virus adaptation experiments were performed. Surprisingly, the conservative I504V change in PB2 evolved again and was the only mutation present in an aggressive virus variant selected during the first adaptation experiment. In contrast, the virulent virus selected in the second adaptation experiment had a lysine-to-arginine change at position 208 in PB1 and a glutamate-to-glycine change at position 349 in PA. These results demonstrate that a variety of minor amino acid changes in the viral polymerase can contribute to enhanced virulence of influenza A virus. Interestingly, all virulence-enhancing mutations that we identified in this study resulted in substantially increased viral polymerase activity.Influenza virus infections continue to represent a major public health threat. Epidemics caused by influenza A viruses (FLUAV) occur regularly, often leading to excess mortality in susceptible populations, and may result in devastating pandemics for humans (37). An avian FLUAV originating from Asia and currently circulating among domestic birds in many countries has the potential to infect and kill people. If further adaptation to humans occurs, this virus strain might become the origin of a future pandemic (57). Although influenza viruses are well characterized, the molecular determinants governing cross-species adaptation and enhanced virulence of emerging virus strains in humans are presently not well understood. The known viral virulence factors are the envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA), the nonstructural proteins NS1 and PB1-F2, and the polymerase complex. HA and NA are of key importance for host specificity and virulence because they determine specific receptor usage and efficient cell entry, as well as formation and release of progeny virus particles. NS1 is a multifunctional protein with interferon-antagonistic activity able to suppress host innate immune responses (11, 15). The small proapoptotic protein PB1-F2 induces more-severe pulmonary immunopathology and increases susceptibility to secondary bacterial pneumonia (3, 30). Recent evidence indicates that the polymerase complex consisting of the three subunits PA, PB1, and PB2 is also a determinant of virulence. Analyses of the 1918 pandemic virus showed that PB1 contributed to the high virulence of this deadly strain (38, 54, 56). Likewise, PB1 also contributed to the unusually high virulence of the pandemic viruses of 1957 and 1968 (23, 47). Interestingly, in recent avian-to-human transmissions of H5N1 and H7N7 viruses, the PB2 subunit was found to play a critical role (32, 40). Molecular studies revealed that an E-to-K exchange at position 627 of PB2 facilitates efficient replication of avian viruses in human cells (24, 33) and determines pathogenicity in mammals (18, 32, 51). Furthermore, recent analyses of highly pathogenic H5N1 viruses demonstrated that PA is involved in high virulence of these avian strains for both avian and mammalian hosts (21, 27).Moderately pathogenic FLUAV strains can be rendered more pathogenic by repeated passages in experimentally infected animals (2, 13, 16, 49, 55). During such adaptations, the evolving viruses frequently seem to acquire virulence-enhancing mutations in the polymerase genes. We recently characterized a virus pair with strikingly different virulences in mice and showed that the virulence-enhancing mutations of the highly virulent strain mapped to the HA, NA, and polymerase genes (13). The two A/Puerto Rico/8/34 (A/PR/8/34) strains are referred to here as high-virulence A/PR/8/34 (hvPR8) and low-virulence A/PR/8/34 (lvPR8). Interestingly, hvPR8 is also highly virulent in mice that carry functional alleles of the Mx1 resistance gene (17), most likely because it replicates rapidly enough to evade the innate immune response of naïve hosts (13).Here, we systematically analyzed which mutations in the three viral polymerase genes contribute to enhanced virulence of hvPR8. We found that two conservative mutations, one in PB2 (I504V) and one in PA (I550L), account for the high-virulence phenotype and that each single mutation considerably increases the activity of the reconstituted polymerase complex. Interestingly, in a new mouse adaptation experiment, the same I504V mutation in PB2 was acquired again by a highly virulent isolate as the only change in the polymerase complex. In contrast, another virulent, mouse-adapted isolate acquired two different mutations in PA and PB1. In this case, the change in PA had a greater impact on both enhanced polymerase activity and enhanced virulence than the mutation in PB1. These data demonstrate that increased polymerase activity contributes to high virus virulence and that human FLUAV have a range of options to achieve this goal.(This work was conducted by Thierry Rolling, Iris Koerner, and Petra Zimmermann in partial fulfillment of the requirements for an M.D. degree from the Medical Faculty [T.R.] or a Ph.D. degree from the Faculty of Biology [I.K. and P.Z.] of the University of Freiburg, Germany.)