Some properties of Photosystem II preparations from the cyanobacterium Synechococcus sp. — presence of an l-amino acid oxidase in Photosystem II complexes from Synechococcus sp.

A highly active O₂-evolving Photosystem II complex has been purified from the cyanobacterium Synechococcus sp., and this complex has been compared with the Photosystem II complex previously isolated from this cyanobacterium (Ohno, T., Satoh, K. and Katoh, S. (1986) Biochim. Biophys. Acta 852, 1–8). Further treatment of the O₂-evolving complex with the detergent sodium taurodesoxycholate resulted in a complex which consisted mainly of the 47 and 40 kDa peptides and which had lost the O₂-evolving activity, but which could still reduce 2,6-dichlorophenolindophenol with 1,5-diphenylcarbazide. Previously, we have shown that a flavoprotein of 49 kDa which has an l-amino acid oxidase activity under certain conditions, is a component of highly active Photosystem II preparations from the cyanobacterium Anacystis nidulans (Pistorius, E.K. and Gau, A.E. (1986) FEBS Lett. 206, 243–248). Based on immunological studies with the antiserum raised against the l-amino acid oxidase protein from A. nidulans, we show that a protein which cross-reacts with this antiserum is present in the highly purified Photosystem II preparations from Synechococcus sp. Moreover, an l-amino acid oxidase activity could also be detected in Photosystem II preparations from Synechococcus sp. The enzyme preferentially oxidizes basic l-amino acids as l-arginine, l-ornithine, 2,3-diamino propionic acid and l-citrulline. In contrast to the enzyme from A. nidulansl-lysine is not oxidized. The here shown presence of an l-amino acid oxidase protein in Photosystem II preparations from Synechococcus sp. is an additional support of our hypothesis that a flavoprotein is a functional component of the water-oxidizing enzyme complex.     

Roles for heme–copper oxidases in extreme high-light and oxidative stress response in the cyanobacterium Synechococcus sp. PCC 7002

The ctaCIDIEI and ctaCIIDIIEII gene clusters that encode heme–copper cytochrome oxidases have been characterized in the marine cyanobacterium Synechococcus sp. PCC 7002 and the inactivation of ctaDI was shown to affect high-light adaptation. In this study, Synechococcus sp. PCC 7002 wild-type, ctaDI, ctaDII, and ctaDI–ctaDII double mutants were grown under extreme high-light and oxidative stress to further assess the roles of cytochrome oxidases in cyanobacteria. Cells of the ctaDI mutant strain barely grew under extreme high-light illumination of 4.5 mE m⁻² s⁻¹, suggesting that CtaDI is required for high-light acclimation in Synechococcus sp. PCC 7002. The ctaDI–ctaDII double mutant cells unexpectedly tolerated extreme high-light intensity, indicating that the disruption of ctaDII gene suppresses the high-light sensitivity phenotype of the ctaDI single mutant. The ctaDII mutant cells also exhibited higher tolerance to the oxidative stress compound, methyl viologen, in the growth media. The ctaDII mutant and the ctaDI–ctaDII double mutant cells had approximately twofold higher levels of superoxide dismutase (SOD) activity, indicating that the disruption of ctaDII gene increased the capacity to decompose active oxygen species. These results suggest that the CtaII cytochrome oxidase may be involved with the oxidative stress response, including the control of SOD expression.     

The Biosynthetic Pathway for Myxol-2′ Fucoside (Myxoxanthophyll) in the Cyanobacterium Synechococcus sp. Strain PCC 7002

Synechococcus sp. strain PCC 7002 produces a variety of carotenoids, which comprise predominantly dicylic β-carotene and two dicyclic xanthophylls, zeaxanthin and synechoxanthin. However, this cyanobacterium also produces a monocyclic myxoxanthophyll, which was identified as myxol-2′ fucoside. Compared to the carotenoid glycosides produced by diverse microorganisms, cyanobacterial myxoxanthophyll and closely related compounds are unusual because they are glycosylated on the 2′-OH rather than on the 1′-OH position of the ψ end of the molecule. In this study, the genes encoding two enzymes that modify the ψ end of myxoxanthophyll in Synechococcus sp. strain PCC 7002 were identified. Mutational and biochemical studies showed that open reading frame SynPCC7002_A2032, renamed cruF, encodes a 1′-hydroxylase and that open reading frame SynPCC7002_A2031, renamed cruG, encodes a 2′-O-glycosyltransferase. The enzymatic activity of CruF was verified by chemical characterization of the carotenoid products synthesized when cruF was expressed in a lycopene-producing strain of Escherichia coli. Database searches showed that homologs of cruF and cruG occur in the genomes of all sequenced cyanobacterial strains that are known to produce myxol or the acylic xanthophyll oscillaxanthin. The genomes of many other bacteria that produce hydroxylated carotenoids but do not contain crtC homologs also contain cruF orthologs. Based upon observable intermediates, a complete biosynthetic pathway for myxoxanthophyll is proposed. This study expands the suite of enzymes available for metabolic engineering of carotenoid biosynthetic pathways for biotechnological applications.A wide variety of organisms produce carotenoid glycosides, which act as natural surfactants, stabilize membranes, and possibly contribute to regulating the permeability of membranes to oxygen (4, 41, 51). The first carotenoid glycosides were isolated from saffron in 1818 (6). However, the structures of the glycosylated carotenoids phleixanthophyll and 4-keto-phleixanthophyll were the first to be completely determined, in 1967, after their isolation from Mycobacterium phlei (15, 34). Tertiary glycosides are relatively rare in nature but seem to be common in carotenoid biosynthesis (34). These include the glycosylated carotenoids of the myxobacteria, which have characteristic C-3′,4′ desaturation and C-1′ glycosylation (18, 19). Acylated carotenoid C-1′-glycosides are broadly distributed among bacteria, including Salinibacter ruber and members of the Chloroflexi and Chlorobi (24, 46, 47).The glycosylated carotenoids of cyanobacteria differ from the examples mentioned above in that glycosylation characteristically occurs on the C-2′-hydroxyl group rather than that at the C-1′ position of the ψ end of myxol (3′,4′-didehydro-1′,2′-dihydro-β,ψ-carotene-3,1′,2′-triol) or oscillol (3,4,3′,4′-tetradehydro-1,2,1′,2′-ψ,ψ-carotene-1,2,1′,2′-tetrol) to form myxoxanthophyll or oscillaxanthin, respectively (16, 17). Myxoxanthophyll is thus far found uniquely in members of the phylum Cyanobacteria, and this compound is named after the synonym for this group of organisms, i.e., Myxophyceae (14, 42). As carotenoids from increasingly diverse bacteria are characterized, the apparent uniqueness of myxoxanthophyll to cyanobacteria will probably not persist. For example, the aglycone myxol occurs in marine flavobacteria, along with saporaxanthin (38, 49). Oscillaxanthin, which was once thought to be unique to the Nostophyceae, was recently identified as the major pigment of three strains of Methylobacterium spp. (20). Moreover, oscillol appears to be a precursor of the glycosylated and acylated carotenoids of Thermomicrobium roseum (52).Several variations on the myxoxanthophyll pathway, which lead to a variety of possible compounds, are known to occur in cyanobacteria. A number of different sugars commonly occur in myxoxanthophyll derivatives found in different strains. Strains of Oscillatoria and Spirulina spp. produce compounds that are chinovosides, fucosides, or methylfucosides (1, 7). Derivatives containing fucose have been found in Nostoc punctiforme strain PCC 73102 and Nostoc sp. strain PCC 7120 (45), whereas myxoxanthophyll dimethylfucoside has been found in Synechocystis sp. strain PCC 6803 (42). Variations in the ring oxidation level of the basal compound, with the addition of a keto group at the C-4 position or of an additional hydroxyl group at the C-2 or C-4 position, may also lead to several related compounds.Myxol is presumably synthesized from lycopene, the acyclic precursor of all carotenoids in cyanobacteria (see Fig. ​Fig.1)1) (27). Because of the occurrence of a β-ionone ring in the final product, monocyclic γ-carotene is also presumed to be an intermediate in this pathway (21, 41). The question of what enzyme is responsible for the formation of the β-ionone ring from the linear ψ end of γ-carotene has been contentious but has recently been resolved. Although CrtL-type lycopene cyclases occur in some cyanobacteria, genes for lycopene cyclases of this family do not occur in the genomes of sequenced cyanobacteria that produce myxoxanthophyll (11, 26). Mohamed and Vermaas reported that the open reading frame (ORF) sll0254 encodes an enzyme thought to be involved simultaneously in the cyclization and ψ-end hydroxylation of lycopene in Synechocystis sp. strain PCC 6803 (31). However, subsequent studies of both Synechococcus sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803 showed that this is not the case (11, 26). Like nearly all other cyanobacteria lacking CrtL homologs, both of these cyanobacteria contain two genes, cruA and cruP, which encode lycopene cyclases (26). A third class of organisms, which so far includes only Synechococcus sp. strains PCC 7942 and PCC 6301, have CrtL and CruP homologs.Open in a separate windowFIG. 1.HPLC elution profiles for pigments from two cyanobacteria. (A) HPLC elution profiles (obtained by the jegpsu method) for pigments extracted from the wild type (solid line) and the slr1293 mutant (dotted line) of Synechocystis sp. strain PCC 6803. (B) HPLC elution profiles (obtained by the jegpsu method) for pigments extracted from the wild type (solid line) and SynPCC7002_A1623 mutant (dotted line) of Synechococcus sp. strain PCC 7002. Peak identities: s, synechoxanthin; md, myxol-2′ dimethylfucoside; mf, myxol-2′ fucoside; z, zeaxanthin; c, cryptoxanthin; e, echinenone; and b, β-carotene.The well-conserved β-hydroxylase CrtR functions in the C-3 hydroxylation of myxoxanthophyll, and CrtR also functions in the synthesis of zeaxanthin, cryptoxanthin, and 3′-hydroxy-echinenone (11, 21, 27, 28). CrtR seems to be responsible for all C-3 hydroxylation reactions in Synechocystis sp. strain PCC 6803, and it is notable that CrtR seems to be extremely highly conserved among cyanobacteria (11, 27). Considerable confusion has existed concerning the remaining reactions of this biosynthetic pathway. Mohamed and Vermaas reported that ORF slr1293 encodes the 3′,4′ desaturase in Synechocystis sp. strain PCC 6803 (30). Furthermore, Mohamed and Vermaas additionally reported that the product of ORF sll0254 plays a dual role as a lycopene cyclase and a mediator of ψ-end hydroxylation during myxoxanthophyll biosynthesis in Synechocystis sp. strain PCC 6803 (31). However, subsequent studies have shown that the sll0254 product and its orthologs, renamed CruE, are carotene desaturases/methyltransferases that participate in the synthesis of the aromatic carotenoid synechoxanthin (12, 13).In this study, we identified two genes, cruF and cruG, which encode the C-1′-hydroxylase and 2′-O-glycosyltransferase, respectively, that are uniquely required for mxyoxanthophyll biosynthesis in Synechococcus sp. strain PCC 7002. Orthologs of these genes are found in all sequenced genomes of cyanobacteria that synthesize myxoxanthophyll. Additionally, in contrast to the data in a previous report (30), we show that ORF slr1293 of Synechocystis sp. strain PCC 6803 and its ortholog SynPCC7002_A2031 in Synechococcus sp. strain PCC 7002 do not play an essential role in myxoxanthophyll biosynthesis.     

Synergistic effect of high-light and low temperature on cell growth of the Δ12 fatty acid desaturase mutant in Synechococcus sp. PCC 7002

The effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Δ12 fatty acid desaturase mutant strains. Under a light intensity of 250 μmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 20–38 °C, but growth was non-exponential below 20 °C and ceased at 12 °C. The Δ12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25–38 °C but grew slowly at 22 °C, and no cell growth took place below 18 °C. Under a very high-light intensity of 2.5 mmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 30–38 °C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Δ12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 °C, but were killed at 30 °C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Δ12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 °C under the normal light intensity of 250 μmol m⁻² s⁻¹. These results suggest that growth of the Δ12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in the antenna size of Photosystem I and Photosystem II in Synechococcus sp. strain PCC 7942 grown in the presence of SANDOZ 9785 — a Photosystem II inhibitor

SANDOZ 9785, also known as BASF 13.338, is a pyridazinone derivative that inhibits Photosystem II (PS II) activity leading to an imbalance in the rate of electron transport through the photosystems. Synechococcus sp. strain PCC 7942 cells grown in the presence of sublethal concentration of SANDOZ 9785 (SAN 9785) for 48 hours exhibited a 20% decrease in Chl a per cell. However, no changes were observed in the content of phycocyanin per cell, the size of the phycobilisomes or in the PS II:PS I ratio. From an estimate of PS II electron transport rate under varying light intensities and spectral qualities and analysis of room temperature Chl a fluorescence induction, it was deduced that growth of Synechococcus PCC 7942 in the presence of SAN 9785 leads to a redistribution of excitation energy in favour of PS II. Though the redistribution appears to be primarily caused by changes affecting the Chl a antenna of PS II, the extent of energetic coupling between phycobilisomes and PS II is also enhanced in SAN 9785 grown Synechococcus PCC 7942 cells. There was a reduction in the effective size of PS I antenna based on measurement of P700 photooxidation kinetics. These results indicate that when PS II is partially inhibited, the structure of photosynthetic apparatus alters to redistribute the excitation energy in favour of PS II so that the efficiency of utilization of light energy by the two photosystems is optimized. Our results suggest that under the conditions used, drastic structural changes are not essential for redistribution of excitation energy between the photosystems.Abbreviations APC Allophycocyanin - Chl a chlorophyll a - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophyenyl)-1,1-dimethyl urea - DCIP 2,6-dichlorophenolindophenol - F_o fluorescence when all the reaction centres are open - f_m fluorescence yield when all the reaction centres are closed - F_v variable chlorophyll fluorescence - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic Acid - I₅₀ concentration that causes 50% inhibition in activity - MV methyl viologen - pBQ para benzoquinone - PBS phycobilisome - PC phycocyanin - PS I, PS II Photosystem I, Photosystem II - P700 reaction centre Chl a of PS I - SAN 9785 SANDOZ 9785 i.e. 4-chloro-5-dimethylamino-2-phenyl-3 (2H) pyridazinone, also known as BASF 13.338     

Restricted capacity for PSI-dependent cyclic electron flow in ΔpetE mutant compromises the ability for acclimation to iron stress in Synechococcus sp. PCC 7942 cells

Exposure of wild type (WT) and plastocyanin coding petE gene deficient mutant (ΔpetE) of Synechococcus cells to low iron growth conditions was accompanied by similar iron-stress induced blue-shift of the main red Chl a absorption peak and a gradual decrease of the Phc/Chl ratio, although ΔpetE mutant was more sensitive when exposed to iron deficient conditions. Despite comparable iron stress induced phenotypic changes, the inactivation of petE gene expression was accompanied with a significant reduction of the growth rates compared to WT cells. To examine the photosynthetic electron fluxes in vivo, far-red light induced P700 redox state transients at 820nm of WT and ΔpetE mutant cells grown under iron sufficient and iron deficient conditions were compared. The extent of the absorbance change (ΔA(820)/A(820)) used for quantitative estimation of photooxidizable P700(+) indicated a 2-fold lower level of P700(+) in ΔpetE compared to WT cells under control conditions. This was accompanied by a 2-fold slower re-reduction rate of P700(+) in the ΔpetE indicating a lower capacity for cyclic electron flow around PSI in the cells lacking plastocyanin. Thermoluminescence (TL) measurements did not reveal significant differences in PSII photochemistry between control WT and ΔpetE cells. However, exposure to iron stress induced a 4.5 times lower level of P700(+), 2-fold faster re-reduction rate of P700(+) and a temperature shift of the TL peak corresponding to S(2)/S(3)Q(B)(-) charge recombination in WT cells. In contrast, the iron-stressed ΔpetE mutant exhibited only a 40% decrease of P700(+) and no significant temperature shift in S(2)/S(3)Q(B)(-) charge recombination. The role of mobile electron carriers in modulating the photosynthetic electron fluxes and physiological acclimation of cyanobacteria to low iron conditions is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Isolation of a Δ6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp.strain PCC 7120

The enzyme ⁶-desaturase is responsible for the conversion of linoleic acid (18:2) to -linolenic acid (18:3). A cyanobacterial gene encoding ⁶-desaturase was cloned by expression of a Synechocystis genomic cosmid library in Anabaena, a cyanobacterium lacking ⁶-desaturase. Expression of the Synechocystis ⁶-desaturase gene in Anabaena resulted in the accumulation of -linolenic acid (GLA) and octadecatetraenoic acid (18:4). The predicted 359 amino acid sequence of the Synechocystis ⁶-desaturase shares limited, but significant, sequence similarity with two other reported desaturases. Analysis of three overlapping cosmids revealed a ¹²-desaturase gene linked to the ⁶-desaturase gene. Expression of Synechocystis ⁶-and ¹²-desaturase in Synechococcus, a cyanobacterium deficient in both desaturases, resulted in the production of linoleic acid and -linolenic acid.     

Functional analysis of the iron-stress induced CP 43′ polypeptide of PS II in the cyanobacterium Synechococcus sp. PCC 7942

Under conditions of iron-stress, the Photosystem II associated chlorophyll a protein complex designated CP 43, which is encoded by the isiA gene, becomes the major pigment-protein complex in Synechococcus sp. PCC 7942. The isiB gene, which is located immediately downstream of isiA, encodes the protein flavodoxin, which can functionally replace ferredoxin under conditions of iron stress. We have constructed two cyanobacterial insertion mutants which are lacking (i) the CP 43 apoprotein (designated isiA ^–) and (ii) flavodoxin (designated isiB ^–). The function of CP 43 was studied by comparing the cell characteristics, PS II functional absorption cross-sections and Chl a fluorescence parameters from the wild-type, isiA ^– and isiB ^– strains grown under iron-stressed conditions. In all strains grown under iron deprivation, the cell number doubling time was maintained despite marked changes in pigment composition and other cell characteristics. This indicates that iron-starved cells remained viable and that their altered phenotype suggests an adequate acclimation to low iron even in absence of CP 43 and/or flavodoxin. Under both iron conditions, no differences were detected between the three strains in the functional absorption crossection of PS II determined from single turnover flash saturation curves of Chl a fluorescence. This demonstrates that CP 43 is not part of the functional light-harvesting antenna for PS II. In the wild-type and the isiB ^– strain grown under iron-deficient conditions, CP 43 was present in the thylakoid membrane as an uncoupled Chl-protein complex. This was indicated by (1) an increase of the yield of prompt Chl a fluorescence (F_o) and (2) the persistence after PS II trap closure of a fast fluorescence decay component showing a maximum at 685 nm.Abbreviations Chl chlorophyll - CP 43, CP 47 and CP 43 Chl a binding protein complexes of indicated molecular mass - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_m and F_m fluorescence when all PS II reaction centers are dosed in dark- and light-acclimated cells, respectively - F_o fluorescence when all PS II reaction centers are open in dark acclimated cells - F_v variable fluorescence after dark acclimation (F_m–F_o)     

Synechocystis sp. PCC 6803 — a useful tool in the study of the genetics of cyanobacteria

The cyanobacterium Synechocystis sp. PCC 6803 was the first phototrophic organism to be fully sequenced. The genomic sequence has revealed the structure of the genome and its gene constituents (3167 genes), as well as the relative map positions of each gene. The functions of nearly half of the genes has been deduced using similarity searches. The genome sequence has also allowed for the implementation of systematic strategies to study gene function and the mechanisms of gene regulation on a genome-wide level. Two genome databases, CyanoBase and CyanoMutants, have been established and act as a central repository for information on gene structure and gene function, respectively. As a result of the genome sequencing and the establishment of these databases, Synechocystis sp. PCC 6803 provides an extremely versatile and easy model to study the genetic systems of photosynthetic organisms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO₃ ^? dehydration and the subsequent fixation of CO₂. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein–protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k _cat of 2.0 × 10⁴ s^?1 and k _cat/K _m of 4.1 × 10⁶ M^?1 s^?1 at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25–35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO₂ analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.     

Structure of the Pyoverdin PVD 2908 – a new Pyoverdin from Pseudomonas sp. 2908

An unknown siderophore (pyoverdin) was isolated from the strain Pseudomonas sp. 2908. The structure of the pyoverdin – called PVD 2908 – was elucidated by spectroscopic methods and degradation studies. Some other siderophores were identified by LC/ESI-MS-screening based on the knowledge of PVD 2908.     

Analysis of a new flavodiiron core structural arrangement in Flv1-ΔFlR protein from Synechocystis sp. PCC6803

Flavodiiron proteins (FDPs) play key roles in biological response mechanisms against oxygen and/or nitric oxide; in particular they are present in oxygenic phototrophs (including cyanobacteria and gymnosperms). Two conserved domains define the core of this family of proteins: a N-terminal metallo-β-lactamase-like domain followed by a C-terminal flavodoxin-like one, containing the catalytic diiron centre and a FMN cofactor, respectively. Members of the FDP family may present extra modules in the C-terminus, and were classified into several classes according to their distribution and composition. The cyanobacterium Synechocystis sp. PCC6803 contains four Class C FDPs (Flv1-4) that include at the C-terminus an additional NAD(P)H:flavin oxidoreductase (FlR) domain. Two of them (Flv3 and Flv4) have the canonical diiron ligands (Class C, Type 1), while the other two (Flv1 and Flv2) present different residues in that region (Class C, Type 2). Most phototrophs, either Bacterial or Eukaryal, contain at least two FDP genes, each encoding for one of those two types. Crystals of the Flv1 two core domains (Flv1-ΔFlR), without the C-terminal NAD(P)H:flavin oxidoreductase extension, were obtained and the structure was determined. Its pseudo diiron site contains non-canonical basic and neutral residues, and showed anion moieties, instead. The presented structure revealed for the first time the structure of the two-domain core of a Class C-Type 2 FDP.     

Correction to: Factors driving adaptive radiation in plants of oceanic islands: a case study from the Juan Fernández Archipelago

Journal of Plant Research - The article Factors driving adaptive radiation in plants of oceanic islands: a case study from the Juan Fernández Archipelago, written by Koji Takayama, Daniel J....     

How to tell green from grey? Towards a methodological framework for evaluating the greening of national tax systems

In this paper, we evaluate four types of indicators that can be used for measuring the greening of a tax system: revenue-based indicators, single tax rates, aggregate tax-rate based indicators and the implicit tax rate on energy. We develop an evaluation framework, introducing two principal evaluation criteria: content validity and comprehensiveness, and four statistical criteria: data availability, comparison over time, international comparability and ease of aggregation. Additional analysis regarding the issue of weighting is carried out for the aggregate tax-rate based indicator. The theoretical and methodological evaluation is supplemented and validated empirically using recent data on the Belgian and Flemish tax system. Finally, conclusions are drawn with regard to the strengths and the weaknesses of the four types of indicators, and recommendations are made for further research.     

Purification and characterization of d(+)-carnitine dehydrogenase from Agrobacterium sp. — a new enzyme of carnitine metabolism

d(+)-Carnitine dehydrogenase from Agrobacterium sp. catalyzes the oxidation of d(+)-carnitine to 3-dehydrocarnitine as initial step of d(+)-carnitine degradation. The NAD⁺-specific, cytosolic enzyme was purified 126-fold to apparent electrophoretic homogeneity by 4 chromatographic steps. The molecular mass of the native enzyme was estimated to be 88 kDa by size-exclusion chromatography. It seems to be composed of 3 identical subunits with a relative molecular mass of 28 kDa as found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and laser-induced mass spectrometry. The isoelectric point was found to be 4.7–5.0. The optimum temperature is 37°C and the optimum pH for the oxidation and the reduction reaction are 9.0–9.5 and 5.5–6.5, respectively. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters and amino terminal sequence. Analogues of d(+)-carnitine (l(−)-carnitine, crotonobetaine, γ-butyrobetaine, carnitine amide, glycine betaine, choline) are competitive inhibitors of d(+)-carnitine oxidation. The equilibrium constant of the reaction of d(+)-carnitine dehydrogenase was determined to be 2.2 × 10⁻¹². The purified d(+)-carnitine dehydrogenase has similar kinetic properties to the l(−)-carnitine dehydrogenase from the same microorganism as well as to l(−)-carnitine dehydrogenases of other bacteria.     

Cobitis pontica sp. nova—a new spined loach species (Cobitidae) from the Bulgarian waters

Spined loaches from the Veleka River (Black Sea coast of Bulgaria), earlier confirmed to represent a separate evolution lineage within a 50-chromosome morph occurring in the Black Sea basin, are described as a new species. This species has the karyotype (2n = 50, NF = 90) similar to one from earlier described C. taurica occurring in the Crimean Peninsula, but differs from it by larger and less numerous spots in the fourth Gambetta’s zone of pigmentation and more anterior position of suborbital spine.