Overexpression and biochemical characterization of a carboxyspermidine dehydrogenase from Agrobacterium fabrum str. C58 and its application to carboxyspermidine production.

BACKGROUND: Carboxyspermidine (C-Spd) is a potentially valuable polyamine carboxylate compound and an excellent building block for spermidine synthesis, which is a critical polyamine with significant implications for human health and longevity. C-Spd can also be used to prepare multivalent cationic lipids and modify nucleoside probes. Because of these positive effects on human health, C-Spd is of considerable interest as a food additive and pharmaceutical target. RESULTS: A putative gene afcasdh from Agrobacterium fabrum str. C58, encoding carboxyspermidine dehydrogenase with C-Spd biosynthesis activity, was synthesized and transformed into Escherichia coli BL21 (DE3) for overexpression. The recombinant AfCASDH was purified and fully characterized. The optimum temperature and pH for the recombinant enzyme were 30 °C and 7.5, respectively. The coupled catalytic strategy of AfCASDH and various NADPH regeneration systems were developed to enhance the efficient production of C-Spd compound. Finally, the maximum titer of C-Spd production successfully achieved 1.82 mmol L-1 with a yield of 91% by optimizing the catalytic conditions. CONCLUSION: A novel AfCASDH from A. fabrum str. C58 was characterized that could catalyze the formation of C-Spd from putrescine and l-aspartate-ß-semialdehyde (L-Asa). A whole-cell catalytic strategy coupled with NADPH regeneration was established successfully for C-Spd biosynthesis for the first time. The coupled system indicated that AfCASDH might provide a feasible method for the industrial production of C-Spd. © 2021 Society of Chemical Industry.

Comparative analysis of two paradigm bacteriophytochromes reveals opposite functionalities in two-component signaling.

Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (Agp1). Whereas Agp1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While Agp1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes suggest the use of light-controllable histidine kinases and phosphatases for optogenetics.

Introduction of glucan synthase into the cytosol in wheat endosperm causes massive maltose accumulation and represses starch synthesis.

We expressed a bacterial glucan synthase (Agrobacterium GlgA) in the cytosol of developing endosperm cells in wheat grains, to discover whether it could generate a glucan from cytosolic ADP-glucose. Transgenic lines had high glucan synthase activity during grain filling, but did not accumulate glucan. Instead, grains accumulated very high concentrations of maltose. They had large volumes during development due to high water content, and very shrivelled grains at maturity. Starch synthesis was severely reduced. We propose that cytosolic glucan synthesized by the glucan synthase was immediately hydrolysed to maltose by cytosolic ß-amylase(s). Maltose accumulation resulted in a high osmotic potential in developing grain, drawing in excess water that stretched the seed coat and pericarp. Loss of water during grain maturation then led to shrinkage when the grains matured. Maltose accumulation is likely to account for the reduced starch synthesis in transgenic grains, through signalling and toxic effects. Using bioinformatics, we identify an isoform of ß-amylase likely to be responsible for maltose accumulation. Removal of this isoform through identification of TILLING mutants or genome editing, combined with co-expression of heterologous glucan synthase and a glucan branching enzyme, may in future enable elevated yields of carbohydrate through simultaneous accumulation of starch and cytosolic glucan.

Characterization and improvement of curdlan produced by a high-yield mutant of Agrobacterium sp. ATCC 31749 based on whole-genome analysis.

Curdlan is a bacterial, water-insoluble, linear homopolysaccharide that has been widely used in the food industry. In this study, genome information of strain CGMCC 11546, a UV-induced high-yield mutant of the model curdlan-producing strain Agrobacterium sp. ATCC 31749, was used to investigate the molecular mechanism of curdlan biosynthesis. The maximum curdlan yield of 47.97 ± 0.57 g/L was obtained from strain CGMCC 11546 by using optimal media containing 60 g/L sucrose, 6 g/L yeast, 2 g/L KH2PO4, 0.4 g/L MgSO4·7H2O, 2 g/L CaCO3, 0.1 g/L FeSO4·7H2O, 0.04 g/L MnSO4, and 0.02 g/L ZnCl2 at 30 °C and 280 rpm after 96 h of fermentation. The gel strength of curdlan was improved by 41 % by knocking out the ß-1,3-glucanase genes exoK and exsH of strain CGMCC 11546. Furthermore, the application of curdlan from the ΔexoK-exsH strain in noodles significantly improved the eating quality of both raw and cooked noodles.

Effects of carbon sources on production and properties of curdlan using Agrobaterium sp. DH-2.

Curdlan has wide potential application in the food and biomedical fields due to its unique thermal gel and biological activity. This study investigated the effect of six sugars including glucose, fructose, lactose, maltose, sucrose and xylose as carbon sources on production and properties of curdlan using Agrobacterium sp. DH-2. The maximum production (38.1 g/L and 37.4 g/L, respectively) and yield (0.58 g curdlan/g sucrose and 0.53 g curdlan/g maltose, respectively) of curdlan were achieved by sucrose and maltose, followed by glucose, fructose, lactose and xylose. Scanning electron micrographs showed that the surface of cells was smooth in strain growth phase, while cells were covered by curdlan matrix acted as a net in the curdlan synthesis phase. The highest glucosyltransferase activity (19.9 U/g biomass) corresponded to the maximum curdlan production using the sucrose medium. The molecular weight and gel strength of curdlan were influenced by the carbon sources. The curdlan from xylose medium resulted in a maximum molecular weight of 1.59 × 106 Da and the highest gel strength of 989.2 g/cm2, while the curdlan from sucrose medium resulted in a lowest molecular weight of 1.10 × 106 Da and gel strength of 672.8 g/cm2. The high molecular weight of curdlan had high gel strength.

Substrate Diversity of L-Threonic Acid Dehydrogenase Homologs.

Despite physiological importance of aldonic sugar acids for living organisms, little is known about metabolic pathways of these compounds. Here, we investigated the functional diversity of homologs of L-threonic acid dehydrogenase (ThrDH; UniProt ID: Q0KBC7), an enzyme composed of two NAD-binding domains (PF14833 and PF03446). Ten ThrDH homologs with different genomic context were studied; seven new enzymatic activities were identified, such as (R)-pantoate dehydrogenase, L-altronic acid dehydrogenase, 6-deoxy-L-talonate dehydrogenase, L-idonic acid dehydrogenase, D-xylonic acid dehydrogenase, D-gluconic acid dehydrogenase, and 2-hydroxy-3-oxopantoate reductase activities. Two associated metabolic pathways were identified: L-idonic acid dehydrogenase was found to be involved in the degradation of L-idonic acid through oxidation/decarboxylation in Agrobacterium radiobacter K84, while 2-hydroxy-3-oxopantoate reductase was found to participate in D-glucarate catabolism through dehydration/cleavage in Ralstonia metallidurans CH34.

A novel way to synthesize pantothenate in bacteria involves ß-alanine synthase present in uracil degradation pathway.

Pantothenate is an indispensable vitamin precursor of the synthesis of coenzyme A (CoA), a key metabolite required in over 100 metabolic reactions. ß-Alanine (ß-ala) is an indispensable component of pantothenate. Due to the metabolic relevance of this pathway, we assumed that orthologous genes for ß-alanine synthesis would be present in the genomes of bacteria, archaea, and eukaryotes. However, comparative genomic studies revealed that orthologous gene replacement and loss of synteny occur at high frequency in panD genes. We have previously reported the atypical plasmid-encoded location of the pantothenate pathway genes panC and panB (two copies) in R. etli CFN42. This study also revealed the unexpected absence of a panD gene encoding the aspartate decarboxylase enzyme (ADC), required for the synthesis of ß-ala. The aim of this study was to identify the source of ß-alanine in Rhizobium etli CFN42. In this study, we present a bioinformatic analysis and an experimental validation demonstrating that the source of ß-ala in this R. etli comes from ß-alanine synthase, the last enzyme of the uracil degradation pathway.

Functional genomic analysis of bacterial lignin degraders: diversity in mechanisms of lignin oxidation and metabolism.

Although several bacterial lignin-oxidising enzymes have been discovered in recent years, it is not yet clear whether different lignin-degrading bacteria use similar mechanisms for lignin oxidation and degradation of lignin fragments. Genome sequences of 13 bacterial lignin-oxidising bacteria, including new genome sequences for Microbacterium phyllosphaerae and Agrobacterium sp., were analysed for the presence of lignin-oxidising enzymes and aromatic degradation gene clusters that could be used to metabolise the products of lignin degradation. Ten bacterial genomes contain DyP-type peroxidases, and ten bacterial strains contain putative multi-copper oxidases (MCOs), both known to have activity for lignin oxidation. Only one strain lacks both MCOs and DyP-type peroxidase genes. Eleven bacterial genomes contain aromatic degradation gene clusters, of which ten contain the central ß-ketoadipate pathway, with variable numbers and types of degradation clusters for other aromatic substrates. Hence, there appear to be diverse metabolic strategies used for lignin oxidation in bacteria, while the ß-ketoadipate pathway appears to be the most common route for aromatic metabolism in lignin-degrading bacteria.

Screening of Processed Foods for Transgenic Proteins from Genetically Engineered Plants Using Targeted Mass Spectrometry.

Screening of food products for the presence of material from genetically engineered (GE) plants is typically done using deoxyribonucleic acid (DNA)-based methods to detect the presence of transgenic DNA. In this study, we have demonstrated the feasibility of using targeted mass spectrometry (MS) to detect a protein expressed by transgenic DNA to confirm the presence of GE plant material in processed foods. Scheduled parallel reaction monitoring (sPRM) was used to detect the enzyme, 5-enolpyruvulshikimate-3-phosphate synthase, from Agrobacterium sp. strain CP4 (CP4 EPSPS), which confers glyphosate tolerance in transgenic crops. Five CP4 EPSPS surrogate peptides and their corresponding retention times identified via data-dependent LC/MS/MS analysis of a glyphosate-tolerant soybean certified reference material, GTS 40-3-2, were used to develop the sPRM assay. The assay was used to screen four soy-based infant formulas, four corn-based cereals, corn tortilla chips, and cornmeal for the presence of CP4 EPSPS. At least four of the five selected surrogate peptides were detected in nine of the products analyzed, suggesting that targeted MS can serve as a complementary analytical method to DNA-based methods for the detection of material from GE plants in processed foods.

Alleviation of chromium toxicity in maize by Fe fortification and chromium tolerant ACC deaminase producing plant growth promoting rhizobacteria.

Chromium (Cr) is becoming a potential pollutant with the passage of time. Higher intake of Cr does not only affect the productivity of crops, but also the quality of food produced in Cr polluted soils. In the past, foliar application of Fe is widely studied regarding their potential to alleviate Cr toxicity. However, limited information is documented regarding the combined use of PGPR and foliar Fe. Therefore, the current study was conducted to screen Cr tolerant PGPR and examine effect of foliar Fe with and without Cr tolerant PGPR under Cr toxicity (50 and 100 mg kg-1) in maize (Zea mays) production. Out of 15, two Cr tolerant PGPR were screened, identified (Agrobacterium fabrum and Leclercia adecarboxylata) and inoculated with 500 µM Fe. Results confirmed that Agrobacterium fabrum + 500 µM Fe performed significantly best in improving dry weight of roots and shoot, plant height, roots and shoot length and plant leaves in maize under Cr toxicity. A significant increase in chlorophyll a (51.5%), b (55.1%) and total (32.5%) validated the effectiveness of A. fabrum + 500 µM Fe to alleviate Cr toxicity. Improvement in intake of N (64.7%), P (70.0 and 183.3%), K (53.8% and 3.40-fold) in leaves and N (25.6 and 122.2%), P (25.6 and 122.2%), K (33.3% and 97.3%) in roots of maize at Cr50 and Cr100 confirmed that combined application of A. fabrum with 500 µM Fe is a more efficacious approach for alleviation of Cr toxicity and fortification of Fe comparative to sole foliar application of 500 µM Fe.

A portable electrochemical immunosensor for highly sensitive point-of-care testing of genetically modified crops.

The wide cultivation of genetically modified (GM) crops has raised concerns on the risks to humans and the environment. 5-enolpyruvylshikimate-3-phosphate synthase isolated from Agrobacterium species strain CP4 (CP4-EPSPS) protein is most widely present in these crops. Therefore the measurement of CP4-EPSPS sensitively in a point-of-care testing (POCT) manner for the screening of transgenic plants is demanded. To date the development of quantitative POCT system has not yet been reported. In presented study, an electrochemical immunosensor towards CP4-EPSPS has been fabricated by integrating a portable bioanalytical device with a disposable screen-printed carbon electrode (SPCE) for POCT of GM crops. The dual-functionalized AuNPs were used as nanoprobes and prepared by simultaneously tagging horseradish peroxidase (HRP) and antibody on AuNPs with an exceptionally simple protocol. The sensitivity of the developed nanoprobe-based immunosensor was 62.5-fold higher than that using HRP-labeled antibody. As a result, the proposed immunosensor using SPCE could detect CP4-EPSPS down to 0.050 ng mL-1 with the linear range of 0.10-10 ng mL-1 within 65 min. In addition, the developed method has been validated with genuine GM crops and the results show a good correlation coefficient of 0.9909 compared with those of a commercial ELISA kit. Therefore, this portable electrochemical immunosensor is suitable for rapid and sensitive detection and provides a convenient and reliable platform for POCT assay.

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.

Desensitizing plant EPSP synthase to glyphosate: Optimized global sequence context accommodates a glycine-to-alanine change in the active site.

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the transfer of a carboxyvinyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate and in plants is the target of the herbicide glyphosate. EPSPSs with high catalytic efficiency and insensitivity to glyphosate are of microbial origin, including the enzyme from Agrobacterium strain CP4, in which insensitivity is conferred by an active site alanine. In the sequence context of plant EPSPSs, alanine in place of glycine at the equivalent position interferes with the binding of both glyphosate and PEP. We show here that iterative optimization of maize EPSPS containing the G101A substitution yielded variants on par with CP4 in terms of catalytic activity in the presence of glyphosate. The improvement relative to G101A alone was entirely due to reduction in Km for PEP from 333 to 18 µm, versus 9.5 µm for native maize EPSPS. A large portion of the reduction in Km was conferred by two down-sizing substitutions (L97C and V332A) within 8 Å of glyphosate, which together reduced Km for PEP to 43 µm Although the original optimization was conducted with maize EPSPS, contextually homologous substitutions conferred similar properties to the EPSPSs of other crops. We also discovered a variant having the known glyphosate-desensitizing substitution P106L plus three additional ones that reduced the Km for PEP from 47 µm, observed with P106L alone, to 10.3 µm The improvements obtained with both Ala101 and Leu106 have implications regarding glyphosate-tolerant crops and weeds.

Variability of CP4 EPSPS expression in genetically engineered soybean (Glycine max L. Merrill).

The expression of the CP4 EPSPS protein in genetically engineered (GE) soybean confers tolerance to the Roundup® family of agricultural herbicides. This study evaluated the variability of CP4 EPSPS expression using an enzyme-linked immunosorbent assay in soybean tissues collected across diverse germplasm and 74 different environments in Argentina, Brazil and the USA. Evaluated material included single and combined (stacked) trait products with other GE traits in entries with cp4 epsps gene at one or two loci. The highest level of CP4 EPSPS was observed in leaf tissues, intermediate in forage and seed, and lowest in root tissues. Varieties with two loci had approximately twice the level of CP4 EPSPS expression compared to one locus entries. Variable and non-directional level of CP4 EPSPS was observed with other factors like genetic background, trait stacking, growing region or season. The maximum and average CP4 EPSPS expression levels in seed provided large margins of exposure (MOE of approximately 4000 and 11,000, respectively), mitigating concerns over exposure to this protein in food and feed from soybean varieties tolerant to Roundup® herbicides.

The crystal structure of XdpB, the bacterial old yellow enzyme, in an FMN-free form.

Old Yellow Enzymes (OYEs) are NAD(P)H dehydrogenases of not fully resolved physiological roles that are widespread among bacteria, plants, and fungi and have a great potential for biotechnological applications. We determined the apo form crystal structure of a member of the OYE class, glycerol trinitrate reductase XdpB, from Agrobacterium bohemicum R89-1 at 2.1 Å resolution. In agreement with the structures of the related bacterial OYEs, the structure revealed the TIM barrel fold with an N-terminal ß-hairpin lid, but surprisingly, the structure did not contain its cofactor FMN. Its putative binding site was occupied by a pentapeptide TTSDN from the C-terminus of a symmetry related molecule. Biochemical experiments confirmed a specific concentration-dependent oligomerization and a low FMN content. The blocking of the FMN binding site can exist in vivo and regulates enzyme activity. Our bioinformatic analysis indicated that a similar self-inhibition could be expected in more OYEs which we designated as subgroup OYE C1. This subgroup is widespread among G-bacteria and can be recognized by the conserved sequence GxxDYP in proximity of the C termini. In proteobacteria, the C1 subgroup OYEs are typically coded in one operon with short-chain dehydrogenase. This operon is controlled by the tetR-like transcriptional regulator. OYEs coded in these operons are unlikely to be involved in the oxidative stress response as the other known members of the OYE family because no upregulation of XdpB was observed after exposing A. bohemicum R89-1 to oxidative stress.

Boosted Electrochemical Immunosensing of Genetically Modified Crop Markers Using Nanobody and Mesoporous Carbon.

The problems of environmental security and the potential risks of human health caused by transgenic crops have attracted much attention. Recent studies reveal 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4 protein (CP4-EPSPS), which shows very high resistance to herbicide glyphosate, is a typical biomarker of genetically modified (GM) crops. For this reason, it is highly anticipated to devise a sensitive and convenient strategy to detect CP4-EPSPS protein in crops. Herein, we report a simple electrochemical immunosensor by coupling nanobody, ordered mesoporous carbon (OMC), and thionine (Th). As a capture agent, the nanobody was screened out from an immunized Bactrian camel, and exhibited superior properties with respect to conventional antibody, such as higher stability and stronger heat resistance. Moreover, OMC offered an effective platform with high surface area, electrical conductivity, and biocompatibility, which greatly facilitated the assembly of redox probe Th, and further coupling of large amount of capture nanobodies. As a result, the CP4-EPSPS protein could be determined with high sensitivity and efficiency by differential pulse voltammetry (DPV) in a wide linear range from 0.001 to 100 ng·mL-1 with a low detection limit of 0.72 pg·mL-1, which was more than 3 orders of magnitude lower than those of previously reported works. As an example, the proposed electrochemical immunosensor was successfully applied to spiked samples, demonstrating its great potential in CP4-EPSPS screening and detection.

Trimethylphosphate and Dimethylphosphate Hydrolysis by Binuclear CdII , MnII , and ZnII -FeII Promiscuous Organophosphate-Degrading Enzyme: Reaction Mechanisms.

Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 exhibits promiscuity, not only in the reactions it catalyzes, but also in the metals it uses to catalyze those reactions. Here, three different binuclear metal centers were studied: di-CdII , di-MnII and ZnII -FeII . This enzyme uses these metal centers for hydrolyzing trimethyl- and dimethyl-phosphates. Both mechanisms were studied at DFT level of theory using a cluster model approach. The ground spin state was determined for each enzyme. The outcomes confirmed that the hydrolysis of phosphotriester is faster than that of phosphodiester and in some case the phosphodiesterase reaction does not occur. The computed activation energies are in agreement with previous experimental results for phosphotriesterase enzymes.

C-5a-substituted validamine type glycosidase inhibitors.

A series of N-alkyl derivatives of the D-galactosidase inhibitor 1,4-di-epi-validamine featuring lipophilic substituents at position C-5a was prepared and screened for their glycosidase inhibitory properties. Products turned out selective for ß-galactosidases as well as ß-glucosidases.

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.