Adsorption Mechanisms of Glyphosate on Ferrihydrite: Effects of Al Substitution and Aggregation State.

Ferrihydrite is one of the most reactive iron (Fe) (oxyhydr)oxides in soils, but the adsorption mechanisms of glyphosate, the most widely used herbicide, on ferrihydrite remain unknown. Here, we determined the adsorption mechanisms of glyphosate on pristine and Al-substituted ferrihydrites with aggregated and dispersed states using macroscopic adsorption experiments, zeta potential, phosphorus K-edge X-ray absorption near-edge structure spectroscopy, in situ attenuated total reflectance Fourier transform infrared spectroscopy coupled with two-dimensional correlation spectroscopy, and multivariate curve resolution analyses. Aggregation of ferrihydrite decreases the glyphosate adsorption capacity. The partial substitution of Al in ferrihydrite inhibits glyphosate adsorption on aggregated ferrihydrite due to the decrease of external specific surface area, while it promotes glyphosate adsorption on dispersed ferrihydrite, which is ascribed to the increase of surface positive charge. Glyphosate predominately forms protonated and deprotonated, depending on the sorption pH, monodentate-mononuclear complexes (MMH1/MMH0, 77-90%) on ferrihydrites, besides minor deprotonated bidentate-binuclear complexes (BBH0, 23-10%). Both Al incorporation and a low pH favor the formation of the BB complex. The adsorbed glyphosate preferentially forms the MM complex on ferrihydrite and preferentially bonds with the Al-OH sites on Al-substituted ferrihydrite. These new insights are expected to be useful in predicting the environmental fate of glyphosate in ferrihydrite-rich environments.

Multiple resistance mechanisms to penoxsulam in Echinochloa crus-galli from China.

Penoxsulam is an important herbicide for the control of Echinochloa crus-galli (L.) P. Beauv. Two resistant populations 17GA (R1) and 16NXB (R2) showed 17- and 3-fold resistance to penoxsulam, respectively. A known resistance mutation of Trp-574-Leu in ALS gene and enhanced rates of penoxsulam metabolism likely involving GST contribute to penoxsulam resistance in R1 population. This population had resistance to the ALS-inhibitors pyribenzoxim and bispyribac­sodium and the auxin herbicide quinclorac, but was susceptible to ACCase-inhibitors quizalofop-p-ethyl and cyhalofop-butyl. No known mutations in the ALS gene conferring target site resistance to ALS-inhibiting herbicides were presented in R2 population. However, penoxsulam metabolism in R2 plants was about 4-fold greater than in susceptible population 14YC (S0) plants. The enzyme inhibitors piperonyl butoxide, malathion and 4-chloro-7-nitrobenzoxadiazole reversed penoxsulam resistance in this population. GST and P450 enzyme activities and the genes of GST1-1, GST1-2, GST1-3, CYP81A18, CYP81A12, CYP81A21 were increased significantly in R2 population. These results indicate that multiple resistance mechanisms had occurred in E. crus-galli populations in central China and resistance needs to be managed effectively by diverse chemical and non-chemical methods.

A highly efficient and cost-effective recombinant production of a bacterial photolyase from the Antarctic isolate Hymenobacter sp. UV11.

Photolyases are DNA-repairing flavoproteins that are represented in most phylogenetic taxa with the exception of placental mammals. These enzymes reduce the ultraviolet-induced DNA damage; thus, they have features that make them very attractive for dermatological or other medical uses, such as the prevention of human skin cancer and actinic keratosis. In this work, we identified a 50.8 kDa photolyase from the UVC-resistant Antarctic bacterium Hymenobacter sp. UV11. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and its activity was analyzed using different approaches: detection of cyclobutane pyrimidine dimers (CPDs) by immunochemistry, high-performance liquid chromatography and comet assays using Chinese Hamster Ovary (CHO) and immortalized nontumorigenic human epidermal (HaCat) cells. The information supports that the recombinant protein has the ability to repair the formation of CPDs, on both double- and single-stranded DNA. This CPD-photolyase was fully active on CHO and HaCat cell lines, suggesting that this enzyme could be used for medical or cosmetic purposes. Results also suggest that the UV11 CPD-photolyase uses MTHF as chromophore in the antenna domain. The potential use of this recombinant enzyme in the development of new inventions with pharmaceutical and cosmetic applications is discussed during this work.

Hormetic Effects of Carbendazim on the Virulence of Botrytis cinerea.

The ascomycete plant-pathogenic fungus Botrytis cinerea infects more than 1,400 plant species worldwide. Stimulatory effects of sublethal doses of fungicides on plant pathogens are of close relevance to disease management. In the present study, stimulatory effects of carbendazim on the virulence of B. cinerea to cucumber plants were investigated. Spraying carbendazim on cucumber plants at 3 to 200 µg/ml had stimulatory effects on the virulence of carbendazim-resistant isolates of B. cinerea and the maximum percent stimulations were 16.7 and 13.5% for isolates HBtom451 and HBstr491, respectively. Preconditioned mycelia (i.e., mycelia grown on potato dextrose agar [PDA] amended with carbendazim at concentrations of 10, 50, or 200 µg/ml) also showed increased virulence, and the maximum percent stimulations for isolates HBtom451 and HBstr491 were 7.9 and 9.5%, respectively. Compared with mycelia grown on PDA without carbendazim, virulence stimulation magnitudes of spraying carbendazim on leaves increased moderately but the concentrations of carbendazim that elicited the maximum stimulation increased 20- and 8-fold for preconditioned isolates HBtom451 and HBstr491, respectively. The time course of infection indicated that virulence stimulation was mediated by a direct stimulation mechanism. Studies of the physiological mechanism for stimulation demonstrated that carbendazim had no significant effects on tolerance to hydrogen peroxide, or on oxalic acid production in B. cinerea. These studies will deepen our understanding of quantitative features of hormetic effects of sublethal doses of fungicides on plant pathogens.

Hormetic Effects of Flusilazole Preconditioning on Mycelial Growth and Virulence of Sclerotinia sclerotiorum.

Hormetic effects of fungicides are highly relevant to fungicide applications and management of plant-pathogenic fungi. Preconditioning (i.e., early exposure to relatively low doses of a toxicant) is a special form of hormesis, and fungicide preconditioning of phytopathogenic fungi is inevitable in the field. The present study showed that spraying the demethylation inhibitor (DMI) fungicide flusilazole at 0.1 µg/ml had stimulatory effects on the virulence of Sclerotinia sclerotiorum inoculated at 1 and 24 h after spraying. Flusilazole sprayed at 10 µg/ml showed inhibitory effects on the virulence of S. sclerotiorum inoculated during the first 3 days after spraying. Inoculations on the 5th, 7th, and 10th day after spraying did not show any significant inhibitory or stimulatory effects on the virulence. After growing for 2 days on potato dextrose agar (PDA) amended with flusilazole at a dose range from 0.0005 to 0.25 µg/ml as preconditioning treatments, mycelia were transferred onto PDA without fungicide and subsequent mycelial growth was slower than the nonpreconditioned control. However, after the preconditioned colonies were transferred onto PDA supplemented with flusilazole at 0.2 µg/ml, percent stimulations of mycelia growth compared with the control had a parabolic shape across the preconditioning flusilazole concentration range. Similarly, the mycelial growth of the preconditioned mycelial plugs on PDA amended with other DMI fungicides (prochloraz or tebuconazole) also showed a typical hormetic response, whereas mycelial growth on PDA amended with carbendazim or dimethachlone was inhibited in a dose-dependent manner. Preconditioning S. sclerotiorum with flusilazole on rapeseed plants elicited virulence stimulations in a dose-dependent manner similar to those on mycelial growth on PDA. After disease lesions developed on rapeseed leaves sprayed with flusilazole as the preconditioning treatment were inoculated onto rapeseed plants, virulence was inhibited on leaves without fungicide or sprayed with carbendazim or dimethachlone compared with the nonpreconditioned control, whereas virulence was stimulated on leaves sprayed with flusilazole, prochloraz, or tebuconazole, and the maximum percent stimulation was 10.2%. These results will advance our understanding of hormetic effects of fungicides and of preconditioning hormesis in particular.

A natural glutathione S-transferase gene GSTU23 confers metabolic resistance to metamifop in Echinochloa crus-galli.

Herbicides are essential for farmers to control weed. However, prolonged use of herbicides has caused facilitated the development of herbicide resistance in weeds. Here, the resistant-E. crus-galli (RL5) was obtained by continuous treatment with metamifop for five generations in paddy fields. RL5 plants showed a 13.7-fold higher resistance to metamifop compared to SL5 plants. Pre-treatment with GST inhibitor (NBD-Cl) significantly increased the susceptibility of RL5 plants to metamifop. Faster metamifop metabolism and higher GST activity in RL5 plants than in SL5 plants were also confirmed, highlighting the role of GST in metabolic resistance. RNA-Seq analysis identified EcGSTU23 as a candidate gene, with up-regulated expression observed in RL5 and field-resistant E. crus-galli plants. Furthermore, the EcGSTU23 gene was overexpressed in the transgenic EcGSTU23-Maize, and the EcGSTU23-Maize showed resistance to metamifop. In vitro metabolic studies also revealed that the purified EcGSTU23 displayed increased catalytic activity in glutathione (GSH) conjugation, and metamifop was metabolized more rapidly in the co-incubation system containing EcGSTU23 protein. These results provide direct experimental evidence of EcGSTU23's involvement in the metabolic resistance of E. crus-galli to metamifop. Understanding the resistance mechanism can help in devising effective strategies to combat herbicide resistance and breeding of genetically modified herbicide resistant crops.

2-{(E)-4-[4-(Trifluoro-meth-yl)phen-oxy]but-2-en-yloxy}phenyl N-methyl-carbamate.

In the title compound, C19H18F3NO4, which was designed and synthesized as a dual-site inhibitor of insect AChE (acetyl-cholinesterase), the dihedral angle between the methyl-carbamate group and the benzene ring is 72.47 (6)°. In the crystal, inversion dimers are linked by pairs of N-H⋯O hydrogen bonds.

Cyhalofop-butyl and pyribenzoxim-induced oxidative stress and transcriptome changes in the muscle of crayfish (Procambarus clarkii).

Cyhalofop-butyl and pyribenzoxim are commonly used herbicides in rice-crayfish co-culture fields. In actual production, weed control in paddy fields is inseparable from cyhalofop-butyl and pyribenzoxim, while its risk to P. clarkii is still unclear. The present study investigated the risk of acute and subchronic toxicity of cyhalofop-butyl and pyribenzoxim to P. clarkii. The results showed that cyhalofop-butyl and pyribenzoxim exposure for 28 days could accumulate in P. clarkii muscle and inhibit P. clarkii growth. Further research found that the malondialdehyde (MDA) level and glutathione-S-transferase (GST) activity in muscle of P. clarkii were significantly increased after exposure to cyhalofop-butyl and pyribenzoxim (4 days and 28 days), and the superoxide dismutase (SOD) and catalase (CAT) activities were significantly altered. Histological results also confirmed cyhalofop-butyl and pyribenzoxim-induced muscle damage in P. clarkii. Additionally, after 28 days exposure to 1.02 mg/L cyhalofop-butyl and 10.4 mg/L pyribenzoxim, transcriptome analysis identified 2029 and 4246 differentially expressed genes (DEGs), respectively. Exposure to 1.02 mg/L cyhalofop-butyl significantly altered metabolism-related pathways, such as drug metabolism-other enzymes, glutathione metabolism, drug metabolism-cytochrome P450, fatty acid biosynthesis and fatty acid degradation. While the pathways related to antioxidant system and nutrient substances synthesis and metabolic were significantly enriched after exposure to 10.4 mg/L pyribenzoxim. This research has significant implications for scientific and rational use of herbicides under rice-crayfish co-culture and will contribute to the development of the highly productive agricultural model.

The complex impacts of economic growth pressure on carbon emission intensity: an empirical evidence from city data in China.

Excessive carbon emissions are the major challenge to global sustainable development. In the context of the coronavirus pandemic, pressure on global economic growth is gradually rising, threatening established carbon reduction targets. However, the relationship between economic growth pressures and carbon emission intensity has yet to be clearly discussed. Thus, this study quantitatively discusses the impacts of economic growth pressures from central (EGPN) and provincial (EGPP) governments on city carbon intensity. The study is based on data from China's city panels from 2005 to 2019. This study finds that (1) there is a U-shaped correlation between economic growth pressure and a city's carbon emission intensity, whether the economic growth pressure comes from the central government or the provincial government; (2) carbon emission intensity is more sensitive to economic growth pressure from the provincial government than it is to economic growth pressure from the central government. The findings of this study will help enhance the understanding of the relationship between economic growth pressure and carbon emission intensity, and can also provide a reference for global sustainable development that balances economic growth and environmental protection.

Metabolic pathways modulated by coumarin to inhibit seed germination and early seedling growth in Eleusine indica.

Coumarin is an allelochemical that is widely present in the plant kingdom and has great potential for weed control. However, its mechanisms of action remain largely unknown. This study employed metabolomic and transcriptomic analyses along with evaluations of amino acid profiles and related physiological indicators to investigate how coumarin inhibits the germination and seedling growth of Eleusine indica by modifying metabolic pathways. At 72 h of germination at 50 and 100 mg L-1 coumarin, E. indica had lower levels of soluble sugar and activities of amylases and higher levels of starch, O2-, H2O2, auxin (IAA) and abscisic acid (ABA) compared to the control. Metabolomic analysis demonstrated that coumarin treatments had a significant impact on the pathways associated with amino acid metabolism and transport and aminoacyl-tRNA biosynthesis. Exposure to coumarin induced significant alterations in the levels of 19 amino acids, with a decrease in 15 of them, including Met, Leu and γ-aminobutyric acid (GABA). Additionally, transcriptomic analysis showed that coumarin significantly disrupted several essential biological processes, including protein translation, secondary metabolite synthesis, and hormone signal transduction. The decrease in TCA cycle metabolite (cis-aconitate, 2-oxoglutarate, and malate) contents was associated with the suppression of transcription for related enzymes. Our findings indicate that the inhibition of germination and growth in E. indica by coumarin involves the suppression of starch conversion to sugars, modification of the amino acid profile, interference of hormone signalling and the induction of oxidative stress. The TCA cycle appears to be one of the most essential pathways affected by coumarin.

Bioaccumulation, metabolism and endocrine-reproductive effects of metolachlor and its S-enantiomer in adult zebrafish (Danio rerio).

The aim of the present study was to evaluate the enantioselective bioaccumulation, metabolism, and toxic effects of metolachlor and S-metolachlor in zebrafish. Five-month-old zebrafish were exposed to metolachlor and S-metolachlor for 28 days, then transferred to clean water and purified for 7 days. In the uptake phase, S-metolachlor was preferentially accumulated at low concentrations, while metolachlor was preferentially accumulated at high concentrations. The two chemicals were metabolized by >70% in zebrafish on the first day and showed same metabolic process. At the accumulation endpoint, S-metolachlor had no significant inhibitory effect on the enzymes activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) and developmental indicators of zebrafish. However, 300 µg/L metolachlor significantly inhibited the enzymes activities of SOD, CAT and GST and affected the liver development. The preferential enrichment of metolachlor at the high concentration may be the reason for its higher toxicity to zebrafish. Further research demonstrated that metolachlor significantly altered the expression of hypothalamic-pituitary-gonadal (HPG) axis-related genes, including gnrh2, gnrh3, lhß, 17ßhsd and cyp19a, thereby reducing the levels of testosterone (T) in females and sex hormones (estradiol and testosterone) in males. S-metolachlor increased the levels of estradiol (E2) in females by altering the expression of HPG axis-related genes such as fshß, cyp17, 17ßhsd and cyp19a. The mechanism of metolachlor and S-metolachlor on the endocrine disrupting effects of zebrafish is different, which may be sex-specific. 7 days after transferring the exposed zebrafish to clean water, most of the enzymes activities, sex hormone levels and related gene expression levels returned to normal, which may be related to the rapid metabolism of the two chemicals.

Metribuzin resistance via enhanced metabolism in a multiple herbicide resistant Lolium rigidum population.

BACKGROUND: The photosystem II (PSII)-inhibiting herbicides are important for Australian farmers to control Lolium rigidum Gaud. and other weed species in trazine tolerant (TT)-canola fields. A L. rigidum population (R) collected from a TT-canola field from Western Australia showed multiple resistance to PSII, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors. The mechanisms of multiple resistance in this R population were determined. RESULTS: The R population showed a low-level (about 3.0-fold) resistance to the PSII-inhibiting herbicides metribuzin and atrazine. Sequencing of the psbA gene revealed no differences between the R and susceptible (S) sequences. Furthermore, [14 C]-metribuzin experiments found no significant difference in metribuzin foliar uptake and translocation between the R and S plants. However, [14 C]-metribuzin metabolism in R plants was 2.3-fold greater than in S plants. The cytochrome P450 monooxygenase inhibitor piperonyl butoxide (PBO) enhanced plant mortality response to metribuzin and atrazine in both R and S populations. In addition, multiple resistance to ALS and ACCase inhibitors are due to known resistance mutations in ALS and ACCase genes. CONCLUSION: The results demonstrate that enhanced metribuzin metabolism likely involving cytochrome P450 monooxygenase contributes to metribuzin resistance in Lolium rigidum. This is the first report of metabolic resistance to the PSII-inhibiting herbicide metribuzin in Australian Lolium rigidum. © 2020 Society of Chemical Industry.

4-Chloro-5-[(5,5-dimethyl-4,5-dihydro-isoxazol-3-yl)sulfonyl-meth-yl]-3-methyl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazole.

The mol-ecule of the title compound, C(12)H(15)ClF(3)N(3)O(3)S, is twisted, as indicated by the C-S-C-C torsion angle of 66.00 (18)° for the atoms linking the ring systems. An intra-molecular C-H⋯F short contact occurs. In the crystal, non-classical C-H⋯O inter-actions, one of which has a short H⋯O contact of 2.28 Å, link the mol-ecules.

4-Bromo-5-[(5,5-dimethyl-4,5-dihydro-isoxazol-3-yl)sulfonyl-meth-yl]-3-methyl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazole.

In the title compound, C(12)H(15)BrF(3)N(3)O(3)S, which has potential herbicidal activity, the mol-ecule is twisted, as indicated by the C-S-C-C torsion angle of 67.86 (19)° for the atoms linking the ring systems. An intra-molecular C-H⋯F short contact occurs and inter-molecular C-H⋯O inter-actions link the mol-ecules in the crystal.

Evidence for weak interaction between phytochromes Agp1 and Agp2 from Agrobacterium fabrum.

During bacterial conjugation, plasmid DNA is transferred from cell to cell. In Agrobacterium fabrum, conjugation is regulated by the phytochrome photoreceptors Agp1 and Agp2. Both contribute equally to this regulation. Agp1 and Agp2 are histidine kinases, but, for Agp2, we found no autophosphorylation activity. A clear autophosphorylation signal, however, was obtained with mutants in which the phosphoaccepting Asp of the C-terminal response regulator domain is replaced. Thus, the Agp2 histidine kinase differs from the classical transphosphorylation pattern. We performed size exclusion, photoconversion, dark reversion, autophosphorylation, chromophore assembly kinetics and fluorescence resonance energy transfer measurements on mixed Agp1/Agp2 samples. These assays pointed to an interaction between both proteins. This could partially explain the coaction of both phytochromes in the cell.

Two aspartate residues close to the lesion binding site of Agrobacterium (6-4) photolyase are required for Mg2+ stimulation of DNA repair.

Prokaryotic (6-4) photolyases branch at the base of the evolution of cryptochromes and photolyases. Prototypical members contain an iron-sulphur cluster which was lost in the evolution of the other groups. In the Agrobacterium (6-4) photolyase PhrB, the repair of DNA lesions containing UV-induced (6-4) pyrimidine dimers is stimulated by Mg2+ . We propose that Mg2+ is required for efficient lesion binding and for charge stabilization after electron transfer from the FADH- chromophore to the DNA lesion. Furthermore, two highly conserved Asp residues close to the DNA-binding site are essential for the effect of Mg2+ . Simulations show that two Mg2+ bind to the region around these residues. On the other hand, DNA repair by eukaryotic (6-4) photolyases is not increased by Mg2+ . In these photolyases, structurally overlapping regions contain no Asp but positively charged Lys or Arg. During the evolution of photolyases, the role of Mg2+ in charge stabilization and enhancement of DNA binding was therefore taken over by a postiviely charged amino acid. Besides PhrB, another prokaryotic (6-4) photolyase from the marine cyanobacterium Prochlorococcus marinus, PromaPL, which contains no iron-sulphur cluster, was also investigated. This photolyase is stimulated by Mg2+ as well. The evolutionary loss of the iron-sulphur cluster due to limiting iron concentrations can occur in a marine environment as a result of iron deprivation. However, the evolutionary replacement of Mg2+ by a positively charged amino acid is unlikely to occur in a marine environment because the concentration of divalent cations in seawater is always sufficient. We therefore assume that this transition could have occurred in a freshwater environment.

Functional role of an unusual tyrosine residue in the electron transfer chain of a prokaryotic (6-4) photolyase.

Cryptochromes and photolyases form a flavoprotein family in which the FAD chromophore undergoes light induced changes of its redox state. During this process, termed photoreduction, electrons flow from the surface via conserved amino acid residues to FAD. The bacterial (6-4) photolyase PhrB belongs to a phylogenetically ancient group. Photoreduction of PhrB differs from the typical pattern because the amino acid of the electron cascade next to FAD is a tyrosine (Tyr391), whereas photolyases and cryptochromes of other groups have a tryptophan as direct electron donor of FAD. Mutagenesis studies have identified Trp342 and Trp390 as essential for charge transfer. Trp342 is located at the periphery of PhrB while Trp390 connects Trp342 and Tyr391. The role of Tyr391, which lies between Trp390 and FAD, is however unclear as its replacement by phenylalanine did not block photoreduction. Experiments reported here, which replace Tyr391 by Ala, show that photoreduction is blocked, underlining the relevance of Tyr/Phe at position 391 and indicating that charge transfer occurs via the triad 391-390-342. This raises the question, why PhrB positions a tyrosine at this location, having a less favourable ionisation potential than tryptophan, which occurs at this position in many proteins of the photolyase/cryptochrome family. Tunnelling matrix calculations show that tyrosine or phenylalanine can be involved in a productive bridged electron transfer between FAD and Trp390, in line with experimental findings. Since replacement of Tyr391 by Trp resulted in loss of FAD and DMRL chromophores, electron transfer cannot be studied experimentally in this mutant, but calculations on a mutant model suggest that Trp might participate in the electron transfer cascade. Charge transfer simulations reveal an unusual stabilization of the positive charge on site 391 compared to other photolyases or cryptochromes. Water molecules near Tyr391 offer a polar environment which stabilizes the positive charge on this site, thereby lowering the energetic barrier intrinsic to tyrosine. This opens a second charge transfer channel in addition to tunnelling through the tyrosine barrier, based on hopping and therefore transient oxidation of Tyr391, which enables a fast charge transfer similar to proteins utilizing a tryptophan-triad. Our results suggest that evolution of the first site of the redox chain has just been possible by tuning the protein structure and environment to manage a downhill hole transfer process from FAD to solvent.

Modulation of DNA Repair Systems in Blind Cavefish during Evolution in Constant Darkness.

How the environment shapes the function and evolution of DNA repair systems is poorly understood. In a comparative study using zebrafish and the Somalian blind cavefish, Phreatichthys andruzzii, we reveal that during evolution for millions of years in continuous darkness, photoreactivation DNA repair function has been lost in P. andruzzii. We demonstrate that this loss results in part from loss-of-function mutations in pivotal DNA-repair genes. Specifically, C-terminal truncations in P. andruzzii DASH and 6-4 photolyase render these proteins predominantly cytoplasmic, with consequent loss in their functionality. In addition, we reveal a general absence of light-, UV-, and ROS-induced expression of P. andruzzii DNA-repair genes. This results from a loss of function of the D-box enhancer element, which coordinates and enhances DNA repair in response to sunlight. Our results point to P. andruzzii being the only species described, apart from placental mammals, that lacks the highly evolutionary conserved photoreactivation function. We predict that in the DNA repair systems of P. andruzzii, we may be witnessing the first stages in a process that previously occurred in the ancestors of placental mammals during the Mesozoic era.

Crystal Structures of Bacterial (6-4) Photolyase Mutants with Impaired DNA Repair Activity.

PhrB from Agrobacterium fabrum is the first prokaryotic photolyase which repairs (6-4) UV DNA photoproducts. The protein harbors three cofactors: the enzymatically active FAD chromophore, a second chromophore, 6,7-dimethyl-8-ribityllumazine (DMRL) and a cubane-type Fe-S cluster. Tyr424 of PhrB is part of the DNA-binding site and could provide an electron link to the Fe-S cluster. The PhrBY424F mutant showed reduced binding of lesion DNA and loss of DNA repair. The mutant PhrBI51W is characterized by the loss of the DMRL chromophore, reduced photoreduction and reduced DNA repair capacity. We have determined the crystal structures of both mutants and found that both mutations only affect local protein environments, whereas the overall fold remained unchanged. The crystal structure of PhrBY424F revealed a water network extending to His366, which are part of the lesion-binding site. The crystal structure of PhrBI51W shows how the bulky Trp leads to structural rearrangements in the DMRL chromophore pocket. Spectral characterizations of PhrBI51W suggest that DMRL serves as an antenna chromophore for photoreduction and DNA repair in the wild type. The energy transfer from DMRL to FAD could represent a phylogenetically ancient process.

Divalent Cations Increase DNA Repair Activities of Bacterial (6-4) Photolyases.

The (6-4) photolyases of the FeS-BCP group can be considered as the most ancient type among the large family of cryptochrome and photolyase flavoproteins. In contrast to other photolyases, they contain an Fe-S cluster of unknown function, a DMRL chromophore, an interdomain loop, which could interact with DNA, and a long C-terminal extension. We compared DNA repair and photoreduction of two members of the FeS-BCP family, Agrobacterium fabrum PhrB and Rhodobacter sphaeroides RsCryB, with a eukaryotic (6-4) photolyase from Ostreococcus, OsCPF, and a member of the class III CPD photolyases, PhrA from A. fabrum. We found that the low DNA repair effectivity of FeS-BCP proteins is largely stimulated by Mg2+ and other divalent cations, whereas no effect of divalent cations was observed in OsCPF and PhrA. The (6-4) repair activity in the presence of Mg2+ is comparable with the repair activities of the other two photolyases. The photoreduction, on the other hand, is negatively affected by Mg2+ in PhrB, but stimulated by Mg2+ in PhrA. A clear relationship of Mg2+ dependency on DNA repair with the evolutionary position conflicts with Mg2+ dependency of photoreduction. We discuss the Mg2+ effect in the context of structural data and DNA binding.