Identification of the prosthetic group of urocanase. The mode of its reaction with sodium borohydride and of its photochemical reactivation.

Urocanase from Pseudomonas putida and from beef liver were isolated by modifying described procedures. Both enzymes were inactivated and labeled on treatment with tritiated sodium borohydride and gave, upon subsequent hydrolysis, a radioactive acid. The previously reported identity of this acid as 2-hydroxybutanoic acid was disproved by several criteria. Other hydroxy acids were also proved to be different from the radioactive acid derived from urocanase. A large portion of the radioactive material from P. putida was found to be nicotinic acid by 1H NMR spectroscopy, gas-liquid chromatography of its methyl ester, and co-crystallization with authentic reference compounds both as the acid and as the hydrazide. A significant portion of the radioactive material derived from beef liver urocanase also co-crystallized with nicotinic acid. Sodium borohydride-treated inactive urocanase was partially reactivated by light. The action spectrum of the photoreactivation showed a maximum at 330 nm. Treatment of urocanase with sodium borodeuteride followed by hydrolysis afforded a sample of nicotinic acid which carried deuterium mainly in position 6. Both the reversible reducibility of urocanase and its action spectrum of photoreactivation suggest that urocanase contains an enzyme-bound nicotinamide nucleotide molecule which is essential for enzymic activity.     

The stoichiometry of the tightly bound NAD+ in urocanase. Separation and characterization of fully active and inhibited forms of the enzyme

1. Urocanase, purified by classical methods [Keul, V., Kaeppeli, F., Ghosh, C., Krebs, T., Robinson, J. A. and Rétey, J. (1979) J. Biol. Chem. 254, 843-851] from Pseudomonas putida was submitted to high-performance liquid chromatography on a TSK-DEAE column. The enzyme was eluted in three resolved peaks (A, B and C) exhibiting specific activities of 3.4 U/mg, 1.85 U/mg and 0.4 U/mg, respectively. 2. The difference spectra of peaks B and A as well as of C and A showed maxima at 330 nm. 3. Irradiation of peaks B and C at 320 nm resulted in an increase of urocanase activity by 45% and 400%, respectively. Peak A could not be photoactivated. Rechromatography of the photoactivated peaks B and C on the TSK-DEAE column confirmed their partial transformation into peak A. 4. Spectroscopic methods for quantitative protein determination were adapted to urocanase. The stoichiometry of bound NAD+/urocanase (form A) was determined to be 1.75 by enzymic analysis of the free NAD+ released upon acid denaturation of the holoenzyme. A similar stoichiometry (1.8-1.9) was found for all three forms (A, B and C) by biosynthetic incorporation of [7-14C]nicotinate into urocanase using a nicotinate auxotrophic mutant of P. putida. 5. Form A of urocanase showed, after treatment with NaBH4 up to 50% inhibition, an elution pattern (TSK-DEAE column) similar to a mixture of forms A, B and C in the approximate ratio of 1:2:1. None of these forms could be photoactivated. 6. We conclude that form A of the urocanase dimer contains two intact NAD+ molecules. In form B one of the two subunits contains an NAD+-nucleophile adduct which is present in both subunits of form C. Full urocanase activity requires intact NAD+ in both subunits. Intact NAD+ can be regenerated from the adduct but not from the reduced form by photolysis. The two subunits of urocanase are independent both in their catalytic activity and in modification reactions.     

Action Spectrum for Photoactivation of the Water-splitting System in Plastids of Intermittently Illuminated Wheat Leaves

The chloroplasts from wheat leaves developed under intermittent illumination (1 ms light + 12 min dark) were able to photoreduce DPIP with DPC as electron donor but unable to photoreduce DPIP with water as electron donor. On exposure of these leaves to continuous light, the Hill activity with water as electron donor was rapidly induced. The photoactivation was sensitive to the treatment with DCMU prior to exposure to continuous light. The action spectrum for the photoactivation showed a sharp band at 680 nm with a distinct shoulder at 650 nm, and was similar to the absorption spectrum of photosytem-2 particles. These data suggest that the electron transfer driven by photosystem 2 is essential for the activation of the water-splitting system in the chloroplasts of intermittently illuminated leaves.     

Thioglycolate, competitive inhibitor of urocanase.

Urocanase was inhibited by thioglycolate, 2-mercaptoethanol, dithioerythritol, and 3-mercaptopropionate. Thioglycolate inhibited competitively at low concentrations (K_i, 0.1 mM) and protected the active site from modification by sulfite. The inhibited enzyme was reactivated by dialysis. A difference spectrum peak of 328 nm for the thioglycolate-urocanase complex compared to the 327 nm absorption maximum of the NAD-thioglycolate adduct. Several nucleophiles are known to inhibit urocanase. We conclude that thioglycolate, as a nucleophilic agent, inhibits by forming an adduct with the tightly bound NAD of urocanase. These results provide indirect evidence that NAD may be the locus of substrate binding in urocanase.     

Photoactivation of Urocanase in Pseudomonas putida: Possible Role in Photoregulation of Histidine Metabolism

Irradiation by near-ultraviolet light of cells or extracts of Pseudomonas putida increased the urocanase activity. Irradiated cells exhibited enhanced catabolic activity on histidine and urocanate.     

Mechanism of reductive photoactivation of enzymes of C4 pathway

Light, besides initiating primary photochemical processes, alters the redox state of soluble components in chloroplast. The present review attempts to cover the mechanism of reductive photoactivation of enzymes of photosynthetic carbon reduction cycle using key enzymes as examples. The reduced soluble components — ferredoxin, thioredoxin and NADPH, in turn, cause the reduction of disulphides to dithiols of chloroplastic enzymes. NADP-malate dehydrogenase is subject to activation by light through changes in NADPH/NADP. The key enzyme of C₄ photosynthesis-PEP carboxylase, though cytosolic, has been shown to be activated by disulphide/sulphhydryl interconversion by reductants generated in light through chloroplast electron transport flow. PyruvateP _i dikinase activity is controlled by the adenylate energy charge. It remains unclear how light controls the activation of cytosolic enzymes.     

Photoactivation of pseudomonad L-tryptophan oxygenase by electron ejection from its substrate, L-tryptophan

Chemically oxidized, catalytically inactive, pseudomonad l-tryptophan-2,3-dioxygenase (EC 1.13.1.12) can be photoactivated aerobically as well as anaerobically by light of wavelength less than 360 nm. The substrate, l-tryptophan, must be present for photoactivation to proceed. In these studies, a CCl₄ filter was used to block light of wavelength less than 265 nm, preventing photolysis of water and the concomitant production of H₂O₂ (known reductant of tryptophan oxygenase). Photoactivation is not inhibited by superoxide dismutase or formate and is only slightly inhibited by catalase. Nonsubstrate analogues of l-tryptophan, 5-fluorotryptophan (binds to the catalytic site), and α-methyltryptophan (binds to the allosteric site), separately or in concert, do not mediate photoactivation, while another substrate, 6-fluorotryptophan, can. Saturation of the allosteric site with α-methyltryptophan increases the extent of photoactivation in the presence of a nonsaturating level of l-tryptophan, indicating that photoactivation is dependent on the extent of saturation of the catalytic site by l-tryptophan. During the time course of photoactivation, catalytic activity increases faster than does the formation of ferroheme enzyme, indicating that the fully reduced enzyme, (ferroheme)₂(Cu⁺)₂, is formed from the fully oxidized enzyme, (ferriheme)₂(Cu²⁺)₂, subsequent to photoactivation. A significant amount of the half-reduced, catalytically active enzyme, (ferriheme)₂(Cu⁺)₂, exists during the time course of photoactivation. We propose that the mechanism by which electrons enter tryptophan oxygenase is via “electron ejection” [T. R. Hopkins and R. Lumry (1972) Photochem. Photobiol.15, 555–566] from a photoexcited l-tryptophan bound at, the catalvtic site.     

A method for the detection of urocanase on polyacrylamide gels and its use with crude cell extracts of Pseudomonas.

A method is described for the specific detection of urocanase activity on polyacrylamide gels. It is dependent upon the reduction of nitro blue tetrazolium by the product of the urocanase reaction using phenazine methosulphate as a coupling agent. The method has been characterized using crude cell extracts of Pseudomonas testosteroni and Pseudomonas putida. After growth of the organisms in histidine-succinate medium each extract shows only one band of urocanase activity. The enzymes from the two species have significantly different electrophoretic mobilities.     

Continuous production of urocanic acid by immobilized Achromobacter liquidum cells

Several microorganisms having higher L -histidine ammonia-lyase activity were immobilized into polyacrylamide gel lattice. The yield of enzyme activity by immobilization was highest in Achromobacter liquidum IAM 1667. As A. liquidum has urocanase activity, the cells were heat-treated at 70°C for 30 min to inactivate the urocanase. Enzymatic properties of the immobilized A. liquidum cells were investigated and compared with those of the intact cells. No difference was observed between the pH activity curve and optimal temperature for the intact and immobilized cells. The permeability of substrate or product through the cell wall was increased by immobilization of the cells. When an aqueous solution of 0.25M L -histidine (pH 9.0) containing 1mM Mg²⁺ was passed through a column packed with the immobilized A. liquidum cells at a flow rate of SV = 0.06 at 37°C, L -histidine was completely converted to urocanic acid. The L -histidine ammonia-lyase activity of the immobilized cell column was stable over 40 days at 37°C. From the effluent of the immobilized cell column, Urocanic acid was easily obtained in a good yield.     

Involvement of Threonine Dehydratase in Biosynthesis of the α-Ketobutyrate Prosthetic Group of Urocanase

Seventeen mutants of Pseudomonas putida that were unable to grow on threonine as nitrogen source owing to a lack of threonine dehydratase were isolated, and all were found to be unable to synthesize active urocanase. Spontaneous revertants selected for urocanase production concomitantly regained threonine dehydratase. Mutants that were unable to utilize urocanate as carbon source were also isolated, and these were defective in urocanase formation but were normal in threonine dehydratase levels. Since alpha-ketobutyrate is the prosthetic group for urocanase, these results are consistent with the proposal that threonine dehydratase is necessary for urocanase prosthetic group biosynthesis. However, the lack of urocanase activity in threonine dehydratase-negative mutants was shown not to be the result of reduced levels of endogenous free alpha-ketobutyrate, nor to the participation of threonine dehydratase in the initiation of urocanase biosynthesis through the conversion of threonyl-tRNA(Thr) to alpha-ketobutyryl-tRNA(Thr). Other alternatives for the participation of threonine dehydratase in urocanase biosynthesis are discussed.     

Imaging of the DNA damage‐induced dynamics of nuclear proteins via nonlinear photoperturbation

Understanding the cellular response to DNA strand breaks is crucial to decipher the mechanisms maintaining the integrity of our genome. We present a novel method to visualize how the mobility of nuclear proteins changes in response to localized DNA damage. DNA strand breaks are induced via nonlinear excitation with femtosecond laser pulses at λ = 1050 nm in a 3D‐confined subnuclear volume. After a time delay of choice, protein mobility within this volume is analysed by two‐photon photoactivation of PA‐GFP fusion proteins at λ = 775 nm. By changing the position of the photoactivation spot with respect to the zone of lesion the influence of chromatin structure and of the distance from damage are investigated. As first applications we demonstrate a locally confined, time‐dependent mobility increase of histone H1.2, and a progressive retardation of the DNA repair factor XRCC1 at damaged sites. This assay can be used to map the response of nuclear proteins to DNA damage in time and space. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effect of Flavin Inhibitors on Photoactivation of Oospores of Phytophthora cactorum

When dark-grown mature oospores of Phytophthora cactorum were activated to germinate by exposure to 5 uW cm^-2nm^-1 of fluorescent light at 20–22°C in the presence of certain flavin inhibitors such as KI, salicylhydroxamic acid and phenylaceric acid at 40. 1. and 0.1 mM respectively, photoactivation and hence subsequent germination of oospores were inhibited without appreciable irreversible effect on oospore viability. Likewise, when applied during the light period, NaN₃ and KCN at 1 mM reduced photoactivation but had a minimal effect on dark reactions. Diphenylamine, an inhibitor of certain carotenoids, had no effect on photoactivation of oospores. The data suggest that the photoreceptor pigment for activation of oospore germination is a flavin.     

Substrate-mediated inactivation of urocanase from Pseudomonas putida. Evidence for an essential sulfhydryl group

Incubation of urocanase from Pseudomonas putida with either its substrate, urocanic acid, or product, 4'(5')-imidazolone-5'(4')-propionic acid, resulted in an oxygen-dependent inhibition of enzyme activity. Coincident with the inactivation was the stoichiometric incorporation of radioactivity from [14C]urocanate into the protein. NAD+ which is required for activity or urocanase was not directly involved in the inactivation process. The inactivation of urocanase was irreversible, could be partially blocked by the competitive inhibitor imidazolepropionate, and involved the modification of a single active-site thiol. The inhibition resulted from oxidative decomposition of 4'(5')-imidazolone-5'(4')-propionate but was not due to the formation of the major degradative product, 4-ketoglutaramate, since this compound was not an irreversible inactivator of urocanase although it did produce some inhibition at high concentrations. A mechanism is presented in which a reactive imine intermediate in the decomposition scheme is subject to nucleophilic attack by an active-site thiol, thereby generating a covalent enzyme--thioaminal adduct. These results emphasize the importance of a catalytic center sulfhydryl group for urocanase activity.     

Enhancement by chloride ions of photoactivation of oxygen evolution in manganese-depleted photosystem II membranes

The Mn cluster that catalyzes photosynthetic oxygen evolution was removed from the photosystem II (PSII) complex by treating PSII membranes with 1.0 mM NH2OH with concomitant inactivation of oxygen evolution. The cluster was reconstituted by incubating the treated membranes with 1.0 mM Mn2+, 20 mM Ca2+, 10 microM 2,6-dichlorophenolindophenol, and Cl- under illumination with continuous or flashing light to restore the oxygen-evolving capacity. This light-dependent activation (photoactivation) of oxygen evolution did not occur to a significant extent at 3 mM Cl-, but markedly accelerated at higher Cl- concentrations without showing a saturation phenomenon even at 1 M Cl-. At 10 mM Cl- only about 10% of the oxygen-evolving activity before NH2OH treatment was restored by 5-min illumination with continuous light, whereas at 600 mM Cl- about 60% of the original activity was recovered. This acceleration resulted from at least two different actions of Cl-: (1) stabilization of the intermediate state involved in the photoactivation process and (2) increase in the quantum yield of photoactivation. The stabilization of the intermediate was saturated at about 150 mM Cl-, whereas the increase in yield did not show saturation. The Cl(-)-induced increase in quantum yield did not involve any changes in the affinity of either Mn2+ binding or Ca2+ binding for photoactivation, but was rather ascribed to a protective effect of Cl- against inhibition of photoactivation by high concentrations of Mn2+. We also found that removal of the extrinsic 33-kDa protein from the PSII complex increased the Cl- requirement for photoactivation.     

Blue Light-Induced In Vivo Absorbance Changes and In Vitro Activation of Nitrate Reductase in a Nitrate-Starved Chlorella Mutant

Illuminating a colorless mutant of Chlorella vulgaris 11h (M125)with blue light caused a reversible photoreduction of b-typecytochrome, i.e., absorbance increases at 423, 525 and 557 nm.This light-induced reduction of cytochrome b was most pronouncedin nitrate-starved cells, which showed some blue light responsesin carbon metabolism, including enhancement of respiration byblue light as reported previously. Prolonged illumination withblue light caused a decrease in the rate of the reduction. The photoactivation of nitrate reductase in the mutant cellswas studied in both cell-free crude extract and purified enzyme.The absorption spectrum of purified enzyme showed three peaksat 423, 525 and 557 nm after the addition of a reductant, indicatingthat the spectrum is that of cytochrome b associated with nitratereductase. Nitrate reductase activity was easily enhanced byblue light illumination after 1 min; red light had no effecton it. The blue light activation of nitrate reductase was notsignificant in growing cells, which showed its high activity. The relationship between the blue light-induced reduction ofcytochrome b and carbon metabolism is discussed. (Received September 30, 1987; Accepted February 9, 1988)     

Light-induced coupling of aqueous-soluble proteins to liposomes formed from carbene-generating phospholipids.

A novel bilayer-forming phospholipid analogue with a photoactivatable carbene-generating head group was synthesized and characterized with respect to molecular structure and light-induced reactivity. N'-(1,2-Dimyristoyl-sn-glycero-3-phosphoethyl)-N-[m-[3- (trifluoromethyl)diazirin-3-yl]phenyl]thiourea (PED) was prepared by thiocarbamoylation of synthetic dimyristoylphosphatidylethanolamine with 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine. PED formed liposomes in aqueous media. Gel to liquid-crystalline transitions occurred at 10.5 degrees C. Neither PED- nor PED/dimyristoylphosphatidylcholine mixed liposomes underwent major structural changes when photoactivated. Liposome sizes, determined by electron microscopy, were not altered upon light exposure. PED combines the advantages of facile synthesis and timed carbene reactivity by photoactivation at wavelengths greater than or equal to 320 nm. Conditions used for PED photoactivation did not inactivate catalytically active or complex-forming proteins. Light-induced binding of aqueous-soluble proteins to PED containing liposomes was attained through photoactivation in the presence of myoglobin, streptavidin, or trypsin. The proteins mentioned were utilized to characterize carbene-initiated ligand coupling. Procedures described establish a new and versatile method for the formation of proteoliposomes.     

Photoactivation of the latent O2-evolving center in chloroplasts isolated from dark-grown spruce seedlings

The latent O₂-evolving center in chloroplasts isolated from spruce [Picea abies (L.) Karst.] seedlings grown in the dark was readily activated by pre-illuminating the chloroplast suspension with weak white light. The photoactivation depended on pH with the optimum at pH 7–8, and was strongly stimulated by ascorbic acid. The optimal stimulation was also obtained at pH 7–8. The temperature dependence of the photoactivation suggested the involvement of some dark reaction in the activation process.     

Urocanic Acid Production by Sporogenous Bacteria

As the results of a screening of several type cultures of bacteria, Bacillus subtilis var. thermophilus was revealed to be the most powerful strain for urocanic acid production. The accumulation of urocanic acid by this bacteria is caused by deamination of L-histidine, and is particularly accelerated in the presence of a component (X-factor) in meat extract. In the decomposition of urocanic acid the optimal pH of urocanase activity is markedly inhibited by the deviation of pH of the culture medium. The histidase of this bacteria is supposed to be a new exo-type enzyme.     

Photomanipulation of antibiotic susceptibility and biofilm formation of <i>Escherichia coli</i> heterologously expressing photoactivated adenylyl cyclase

A cyaA-deficient Escherichia coli strain was transformed by a plasmid carrying the gene for BsPAC, a photoactivated adenylyl cyclase identified from a Beggiatoa sp., and was subjected to an antibiotic susceptibility assay and biofilm formation assay under a light or dark condition. Cells expressing BsPAC that were incubated under blue light (470 nm) were more susceptible to fosfomycin, nalidixic acid and streptomycin than were cells incubated in the dark. Cells expressing BsPAC formed more biofilms when incubated under the light than did cells cultured in the dark. We concluded from these observations that it is possible to determine the importance of cAMP in antibiotic susceptibility and biofilm formation of E. coli by photomanipulating the cellular cAMP level by the use of BsPAC. A site-directed mutant of BsPAC in which Tyr⁷ was replaced by Phe functioned even in the dark, indicating that Tyr⁷ plays an important role in photoactivation of BsPAC. Results of mutational analysis of BsPAC should contribute to an understanding of the molecular basis for photoactivation of the protein.     

Manganese and calcium requirements for reconstitution of oxygen-evolution activity in manganese-depleted photosystem II membranes

The Mn complex of photosystem II and O2-evolution activity are reconstituted in Mn-depleted photosystem II membranes in a light-dependent process called photoactivation. Recovery of O2-evolution activity requires both Mn2+ and Ca2+ in the photoactivation medium. The Mn2+ and Ca2+ dependences of both the effective rate constant and yield of photoactivation have been determined. A comparison of these data with the predictions of mathematical models for photoactivation leads to the conclusion that photoactivation occurs in two stages. The first stage, photoligation of Mn, requires light and depends primarily on Mn2+. The second stage, binding of Ca2+, is required for expression of O2-evolution activity. This two-stage model affords an excellent fit to the data and provides dissociation constants and binding stoichiometries for Ca2+ and Mn2+. We conclude that one Mn2+ ion is bound and photooxidized in the rate-determining step(s) of photoactivation. On the basis of these results and data already in the literature, the molecular details of the elementary steps in photoactivation are discussed and a mechanism of photoactivation is proposed.