DETECTION OF PTERINS IN Phycomyces SPORANGIOPHORES*

Differential fluorometry with sodium dithionite showed pterin-like signals in extracts of Phycomyces sporangiophores. After iodine oxidation, pterin, biopterin and neopterin could be separated. The concentrations determined for these three pterins exceed the calculated minimal concentration of 3 times 10^?7M for the photoreceptor.     

Altered pterin patterns in photobehavioral mutants of Phycomyces blakesleeanus.

Pterins were extracted with methanol from sporangiophores of the lower fungus Phycomyces blakesleeanus and separated and identified by high performance liquid chromatography (HPLC) with fluorescence detection. The following pterins were found and identified for the wild-type strain NRRL1555: carboxypterin (6.7 x 10(-6) M), neopterin (4.2 x 10(-7) M), xanthopterin (5.3 x 10(-6) M), biopterin (3.9 x 10(-7) M), pterin (9.1 x 10(-7) M), and 6,7-dimethylpterin (1.2 x 10(-6) M). The HPLC elution profiles of the wild type were compared to a set of phototropism mutants (genotype mad) with specific defects in the light-transduction pathway. The mutant profiles were qualitatively similar to those of the wild type. Quantitative differences were, however, discerned for madA, madC, and madH mutants. The madA mutation was associated with increased amounts of biopterin and 6,7-dimethylpterin and a reduction of neopterin, pterin, xanthopterin, and unidentified pterins eluting at 14-18 min. The stimulatory effect of the madA mutation on biopterin and 6,7-dimethylpterin appears to be compensated by a secondary mutation (pde) which is responsible for the loss of 75% of adenosine 3',5'-cyclic monophosphate (cAMP)-phosphodiesterase activity. In a madA pde double mutant the amounts of biopterin and 6,7-dimethylpterin fell below the wild-type level. These results suggest that an increased level of endogenous cAMP represses the biosynthesis of these pterins. The madC mutation increased the amounts of biopterin and xanthopterin and that of the unidentified pterins which could be derivatized to carboxypterin. Single madB mutations had, compared to the wild type, two times higher amounts of biopterin and two times lower amounts of neopterin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Photoreceptor for Phototaxis in the Photosynthetic Flagellate Euglena gracilis

The unicellular flagellate Euglena gracilis shows positive phototaxis at low fluence rates (≤10 W m ²) and negative phototaxis at high fluence rates (≥100 W m ²). Currently, retinal or flavins/pterins are discussed as chromo-phores of the photoreceptor. When grown in the presence of 4 mM nicotine, a retinal inhibitor, for several generations, the cells still showed both responses, indicating that retinal is unlikely to be the chromophoric group of the photoreceptor responsible for phototaxis. The native flavin(s) can be substituted by growing the cells in roseo-flavin dissolved in the medium. The absorption spectrum of roseoflavin extends well beyond the action spectrum for phototaxis (up to 600 nm). Excitation at wavelengths >550 nm does not cause phototactic orientation in control cells but causes both positive and negative phototaxis in roseoflavin-grown cells, indicating an uptake and assembly of the chromophore in the photoreceptor complex. The white mutant strain 1224-5/1f, induced by streptomycin treatment, lacks flavins as indicated by fluorescence spectroscopy. The phototaxis-deficient pheno-type cannot be complemented by the addition of external riboflavin. Fluorescence spectra of intact paraxonemal bodies (PAB) indicate that both pterins and flavins are involved in photoperception and that the excitation energy is efficiently funneled from the pterins to the flavins. This energy transfer is disrupted by solubilization of the PAB. In intact PAB flavins are not accessible to reducing or oxidizing substances, indicating that they are located inside the structure, while pterins are accessible, so that their localization can be assumed to be on the surface. The results described above are discussed with regard to the potential involvement of flavins and pterins as well as retinal in photoperception.     

Enantiomeric separation of biopterin and neopterin stereoisomers by chiral HPLC. Application to the identification of natural pterins in protozoans

Summary The separation of the D and L enantiomers of biopterin and neopterin has been achieved by ligand exchange chromatography using a reversed phase column with a mobile phase containing D-phenylalanine as chiral modifier and Cu(II) as the metal ion. This successful separation has enabled the identification of natural pterins present in various protozoans by comparison with authentic, optically pure compounds. D-monapterin and D-neopterin were identified in the ciliate protozoanColpidium campylum. L-biopterin has been found, for the firs time, in a flagellate protozoan,Astasia longa.     

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 PhrB_Y424F mutant showed reduced binding of lesion DNA and loss of DNA repair. The mutant PhrB_I51W 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 PhrB_Y424F revealed a water network extending to His366, which are part of the lesion‐binding site. The crystal structure of PhrB_I51W shows how the bulky Trp leads to structural rearrangements in the DMRL chromophore pocket. Spectral characterizations of PhrB_I51W 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.     

蝶呤类化合物的荧光性能研究

研究了蝶呤类化合物的天然荧光特性。着重考察了新蝶呤、生物蝶呤、黄蝶呤和蝶呤在 p H7.7磷酸盐缓冲溶液条件下的荧光光谱及各种因素对其荧光强度的影响。在最佳实验条件下 ,四种蝶呤类化合物的线性范围为 :蝶呤 0 .6～ 2 .8μg/m L,新蝶呤 0～ 2 .6μg/m L,生物蝶呤 0～ 2 .4μg/m L,黄蝶呤 0～ 6.0 μg/m L,检出限依次为 :4.2 9× 1 0 - 7g/m L,6.71× 1 0 - 8g/m L,5.79× 1 0 - 9g/m L和 1 .75× 1 0 - 8g/m L     

Analysis of Pterins in Urine by HILIC

Pterins are a class of compounds excreted in urine. Levels of the pterins are found to be significantly elevated in a variety of diseases. A new method involving hydrophilic interaction chromatography with fluorescence detection has been developed for analysis of neopterin, biopterin, and isoxanthopterin in urine samples. Separation of these pterins on an aminopropyl hydrophilic interaction column was achieved by isocratic elution. The effects of the organic modifier content, ionic strength, and pH of the mobile phase on the hydrophilic behavior of the pterins were studied and the mechanism of their separation was also investigated. Under the optimum chromatographic conditions the linearity (r ≥ 0.9995) and repeatability (relative standard deviation < 4.0%) of the method are good. Compared with reversed-phase high-performance liquid chromatography, the method is simple and convenient. The method was applied to the analysis of pterins in urine samples with satisfactory results.     

PTERIN- AND FLAVIN-LIKE FLUORESCENCE ASSOCIATED WITH ISOLATED FLAGELLA OF Euglena gracilis

Fluorescence excitation- and emission spectra indicate the presence of pterin(s) and flavin(s) in isolated flagella of the phytoflagellate Euglena gracilis. These compounds appear to bind at least in part non-covalently to the molecular framework of the paraflagellar body, which is the presumed photoreceptor organelle and which is attached to the isolated flagella. A compound with pterin-like fluorescence excitation and emission spectrum could he extracted with methanol from isolated flagella and could he recovered on thin-layer silica gels. Besides the previously assumed photoreceptor function of flavins, our results suggest also a role for pterins in the photosensory transduction chain of Euglena gracilis.     

A FLAVOPROTEIN MAY MEDIATE THE BLUE LIGHT-ACTIVATED BINDING OF GUANOSINE 5'-TRIPHOSPHATE TO ISOLATED PLASMA MEMBRANES OF Pisum sativum L.*

Abstract— A blue light photoreceptor has not been identified in higher plants. Most proposals for a blue light-absorbing chromophore lack evidence for a direct connection between the putative chromophdre and a biological effect. Fluorescence data for the plasma membrane from etiolated buds of Pisum sativum L. suggest that we are measuring fluorescence emission of flavin species, and probably not pterin species. Fluorescence data indicate that a putative flavin exists associated with a protein or protein complex in the plasma membrane. Excitation of plasma membranes that were boiled in the presence of 0.1% sodium dodecyl sulfate and treated with blue light yields a fluorescence band with a maximum of approximately 552 nm. This fluorescence emission can be rapidly quenched by the flavin antagonists phenylacetic acid (PAA) and KI. Blue light-enhanced binding of guanosine 5'-[Γ-thio]triphosphate (GTPγS) to a protein in the plasma membrane is strongly inhibited by PAA, KI, and NaN₃, all quenchers of flavin excited states, indicating that a chromophore for this photoreaction may be a flavin associated with a plasma membrane protein. The above evidence is consistent with the participation of a flavin as the chromophore for the light-induced GTP-binding reaction in pea plasma membrane.     

BLUE AND ULTRAVIOLET-B LIGHT PHOTORECEPTORS IN PARSLEY CELLS

Abstract— Ultraviolet-B (UV-B) and blue light photoreceptors have been shown to regulate chalcone synthase and flavonoid synthesis in parsley cell cultures. These photoreceptors have not yet been identified. In the present work, we studied UV-B photoreception with physiological experiments involving temperature shifts and examined the possible role of flavin in blue and UV-B light photoreception. Cells irradiated with UV-B light (0.5–15 min) at 2°C have the same fluence requirement for chalcone synthase and flavonoid induction as controls irradiated at 25°C. This is indicative of a purely photochemical reaction. Cells fed with riboflavin and irradiated with 6 h of UV-containing white light synthesize higher levels of chalcone synthase and flavonoid than unfed controls. This effect did not occur with blue light. These results indicate that flavin-sensitization requires excitation of flavin and the UV-B light photoreceptor. The in vivo kinetics of flavin uptake and bleaching indicate that the added flavin may act at the surface of the plasma membrane. In view of the likely role of membrane-associated flavin in photoreception, we measured in vitro flavin binding to microsomal membranes. At least one microsomal flavin binding site was solubilized by resuspension of a microsomal pellet in buffer with high KPi and NaCl concentrations and centrifugation at 38000 g. The 38000 g insoluble fraction had much greater flavin binding and contained a receptor with an apparent K_D of about 3.6 μM and an estimated in vivo concentration of at least 6.7 × 10–⁸M. Flavin mononucleotide, roseoflavin, and flavin adenine dinucleotide can compete with riboflavin for this binding site(s), although each has lower affinity than riboflavin. Most microsomal protein was solubilized by resuspension of the microsomal pellet in non-denaturing detergents and centrifugation at 38 000 g ; however, this inhibited flavin binding, presumably because of disruption of the environment of the flavin receptor. The parsley microsomal flavin binding receptor(s) have a possible role in physiological photoreception.     

PHOTORECEPTOR PROTEINS AND PIGMENTS IN THE PARAFLAGELLAR BODY OF THE FLAGELLATE Euglena gracilis

Abstract— –The presumed photoreceptor for phototaxis, the paraflagellar body, in the flagellate Euglena gracilis , was isolated still attached to the flagellum. After solubilization, fast protein liquid chromatography (FPLC) analysis yielded four major protein fractions with the chromophoric groups still attached. Fluorescence spectra showed that three fractions had excitation peaks at 380 nm and emission peaks around 450 nm indicative of pterins, while the fourth chromoprotein had a fluorescence emission at 520 nm and an excitation peak at 450 nm, indicative of a flavin. The separated proteins were analyzed by gel electrophoresis: the pterin binding proteins have apparent molecular masses between 27 000 and 31 600 and the flavin binding protein has an apparent molecular mass of 33 500.     

Determination of total oncopterin,neopterin and biopterin in human urine by high performance liquid chromatography with solid phase extraction

A new, sensitive method for the determination of oncopterin, biopterin, and neopterin in human urine has been developed using SPE with 6,7‐dimethylpterin as internal standard and gradient HPLC with fluorescence detection. SPE was tested for the pre‐treatment of urine samples on different types of sorbents (strong ion exchange resins, polar adsorbents, and reversed‐phase sorbents). RP‐SPE with subsequent evaporation of eluate has been found to be the most appropriate. The extraction efficiency exceeded 95% for all selected pterins. The extracted pterins were subsequently analyzed on a Purospher RP‐18 RP column. The LOD of oncopterin was 1.43 nmol/L of urine. The intra‐day and inter‐day imprecision at a physiological oncopterin concentration never exceeded 10%. The potential of this method was tested using urine samples of healthy volunteers and cancer patients without methotrexate therapy.     

In‐Planta Expression: Searching for the Genuine Chromophores of Cryptochrome‐3 from Arabidopsis thaliana

Göbel et al. present in this issue an exemplary study of identification of chromophores from Arabidopsis thaliana cryptochrome‐3. Usually taken for granted, proteins and cofactors, respective chromophores, from heterologous expression are considered identical to material isolated from their genuine host. Cryptochromes carry two chromophores, an antenna cofactor and a functional flavin chromophore, both noncovalently embedded into the protein. In particular the antenna chromophore is loosely bound and often lost during protein purification. The authors identify from plant‐extracted Cry3 unambiguously N⁵,N¹⁰‐methenyltetrahydrofolate as antenna chromophore and flavin adenine dinucleotide as the functional chromophore.     

A novel prokaryotic UVB photoreceptor in the cyanobacterium Chlorogloeopsis PCC 6912

We present evidence for the presence and nature of a UVB-specific photoreceptor in the cyanobacterium Chlorogloeopsis PCC 6912. The photoreceptor mediates at least the photosensory induction of mycosporine-like amino acid (MAA) synthesis. Because MAA synthesis in this organism can also be induced under salt stress, we could distinguish between the photosensory and the purely biochemical requirements of MAA synthesis. Neither visible light nor UV radiation was necessary for the biosynthetic process, thus indicating that the UVB (280-320 nm) dependence of biosynthesis is based on a UV photosensory capacity of the organism. An action spectrum of the MAA synthesis showed a distinct peak at 310 nm tailing down into the UVA (320-400 nm) region with no detected activity above 340 nm. We found that radiation below 300 nm caused significant inhibition of synthesis of MAAs indicating that the action spectrum at these wavelengths may not have been satisfactorily resolved. We propose that a pterin is a good candidate for a photoreceptor chromophore as (1) reduced pterins present absorption spectra congruent with the action spectrum obtained; and (2) an inhibitor of the biosynthetic pathway of pterins and an antagonist of excited states of pterins, both depressed the photosensory efficiency of induction but not its chemosensory efficiency.     

DETAILED ACTION SPECTRUM FOR THE DELAY SHIFT IN PUPAE EMERGENCE OF DROSOPHILA PSEUDOOBSCURA*

Abstract— Dose-response curves were measured for the delay phase shift of pupae emergence of Drosophila pseudoobscura. From these curves an action spectrum was determined for this process; it suggests a flavin to be the photoreceptor pigment.     

Photooxidation of Tetrahydrobiopterin under UV Irradiation: Possible Pathways and Mechanisms

Tetrahydrobiopterin (H₄Bip) is a cofactor for several key enzymes, including NO synthases and aromatic amino acid hydroxylases (AAHs). Normal functioning of the H₄Bip regeneration cycle is extremely important for the work of AAHs. Oxidized pterins may accumulate if the H₄Bip regeneration cycle is disrupted or if H₄Bip autoxidation occurs. These oxidized pterins can photosensitize the production of singlet molecular oxygen ¹O₂ and thus cause oxidative stress. In this context, we studied the photooxidation of H₄Bip in phosphate buffer at pH 7.2. We found that UV irradiation of H₄Bip affected its oxidation rate (quantum yield Φ₃₀₀ = (2.7 ± 0.4) × 10^?3). The effect of UV irradiation at λ = 350 nm on H₄Bip oxidation was stronger, especially in the presence of biopterin (Bip) (Φ₃₅₀ = (9.7 ± 1.5) × 10^?3). We showed that the rate of H₄Bip oxidation linearly depends on Bip concentration. Experiments with KI, a selective quencher of triplet pterins at micromolar concentrations, demonstrated that the oxidation is sensitized by the triplet state biopterin ³Bip. Apparently, electron transfer sensitization (Type‐I mechanism) is dominant. Energy transfer (Type‐II mechanism) and singlet oxygen generation play only a secondary role. The mechanisms of H₄Bip photooxidation and their biological meaning are discussed.     

Altered flavin patterns in photobehavioral mutants of Phycomyces blakesleeanus.

Flavins were extracted from sporangiophores of the lower fungus Phycomyces blakesleeanus and identified by HPLC with fluorescence detection. In the wild-type strain NRRL1555 they were found to be present at the following concentrations: riboflavin (5.5 x 10(-6) M), flavin mononucleotide (FMN) (4.0 x 10(-6) M) and flavin adenine dinucleotide (1.4 x 10(-6) M). The HPLC elution profiles of the wild type were compared to a set of behavioral mutants (genotype mad) with specific defects in their light-transduction pathway. The photoreceptor mutants C109 (madB), C111 (madB) and L1 (madC) had normal amounts of flavins. The most prominent changes were found in single mutants with a defective madA gene which contained about 25% of riboflavin and about 10% of FMN and FAD normally found in the wild type. A hypertropic mutant with a defective madH gene contained instead 80% of riboflavin and 120% of FMN and FAD. The double mutant L52 (madA madC) and the triple mutant L72 (madA madB madC) had normal amounts of FAD and FMN. This indicates that the madC mutation, which itself causes loss of light sensitivity and which affects the near-UV/blue-light receptor (Galland and Lipson, 1985, Photochem. Photobiol. 41, 331-335) functions as a restorer of the flavin content in a genetic madA background. The double mutant L51 (madA madB) had about 40% of FMN and FAD, suggesting that the madB mutation functions as a partial restorer of flavin content. The photogravitropic thresholds (450 nm) reported for the wild type and the madA and madH mutants were positively correlated to the endogeneous concentrations of FMN and FAD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pterin Chemistry. Part 92.. Loading experiments with 6α,β-tetrahydro-L-[3′-2H1] biopterin

6α,β-Tetrahydro-L -[3′-²H₁]biopterin ([3′-²H₁]- 1 ) was administered orally to two primapterinuric patients in order to investigate the biosynthetic pathway of 7-substituted pterins in humans. L -Primapterin ( 2 ) and L -biopterin were isolated from urine after loading and measured by GC/MS. L -Biopterin and L -primapterin were labelled with ²H to an equal extent. From this result, one can conclude that L -primapterin is formed from tetrahydro-L -biopterin, very probably via an intramolecular rearrangement.     

Pterin chemistry. Part 88. A simple synthesis of 5-Deoxy-L-[5-2H1]arabinose and L-[3′-2H1] biopterin

A simple synthesis of 5-deoxy-L -[5-²H₁]arabinose was performed to obtain L -[3′-²H₁]biopterin. The reduced form of this model substance is needed to investigate the pathway of 7-substituted pterins in patients with primapterinuria.     

BLUF Hydrogen network dynamics and UV/Vis spectra: A combined molecular dynamics and quantum chemical study

Blue light sensing using flavin (BLUF) protein photoreceptor domains change their hydrogen bond network after photoexcitation. To explore this phenomenon, BLUF domains from R. sphaeroides were simulated using Amber99 molecular dynamics (MD). Five starting configurations were considered, to study different BLUF proteins (AppA/BlrB), Trp conformations (“W_in”/“W_out”), structure determination (X‐ray/NMR), and finally, His protonation states. We found dependencies of the hydrogen bonds on almost all parameters. Our data show an especially strong correlation of the Trp position and hydrogen bonds involving Gln63. The latter is in some contradiction to earlier results (Obanayama et al., Photochem. Photobiol. 2008, 84 10031010). Possible origins and implications are discussed. Our calculations support conjectures that Gln63 is more flexible with Trp104 in W_in position. Using snapshots from MD and time‐dependent density functional theory, UV/vis spectra for the chromophore were determined, which account for molecular motion of the protein under ambient conditions. In accord with experiment, it is found that the UV/vis spectra of BLUF bound flavin are red‐shifted and thermally broadened for all calculated π → π* transitions, relative to gas phase flavin at T = 0 K. However, differences in the spectra between the various BLUF configurations cannot be resolved with the present approach. © 2012 Wiley Periodicals, Inc.