Transformation of 2,2′-Bimorphine to the Novel Compounds 10-α-S-Monohydroxy-2,2′-Bimorphine and 10,10′-α,α′-S,S′-Dihydroxy-2,2′-Bimorphine by Cylindrocarpon didymum

Whole-cell suspensions of Cylindrocarpon didymum were observed to transform 2,2′-bimorphine to the compounds 10-α-S-monohydroxy-2,2′-bimorphine and 10,10′-α,α′-S,S′-dihydroxy-2,2′-bimorphine. Mass spectrometry and ¹H nuclear magnetic resonance spectroscopy confirmed the identities of these new morphine alkaloids.     

Oxidation of Morphine to 2,2′-Bimorphine by Cylindrocarpon didymum

The oxidation of morphine by whole-cell suspensions and cell extracts of Cylindrocarpon didymum gave rise to the formation of 2,2′-bimorphine. The identity of 2,2′-bimorphine was confirmed by mass spectrometry and ¹H nuclear magnetic resonance spectroscopy. C. didymum also displayed activity with the morphine analogs hydromorphone, 6-acetylmorphine, and dihydromorphine, but not codeine or diamorphine, suggesting that a phenolic group at C-3 is essential for activity.The morphine alkaloids are the major alkaloid components of opium, the dried latex material from cut seed capsules of the opium poppy, Papaver somniferum. Of all the alkaloids, the morphine alkaloid group has been studied in most detail, mainly due to the significant therapeutic properties these compounds possess. The morphine alkaloids are narcotic analgesics and are widely used by clinicians for the control of chronic pain. The use of microbial enzymes to provide biological routes for the synthesis of semisynthetic drugs that are difficult to synthesize chemically and as a means of producing new morphine alkaloid derivatives has been the subject of a significant amount of research; this topic has recently been reviewed (3). In recent years, there has been an increasing demand for new morphine alkaloid intermediates for the synthesis of novel semisynthetic drugs, and as part of a study to produce such compounds, we have been exploring fungal transformations of morphine. In this paper, we describe the conversion of morphine to pseudomorphine (2,2′-bimorphine) by Cylindrocarpon didymum 311186.

Biotransformation of morphine.

C. didymum 311186 was obtained from the International Mycological Institute (Egham, Surrey, United Kingdom). Mycelia were grown in media at pH 7.0 containing (grams per liter) yeast extract (10.0), KH₂PO₄ (10.0), (NH₄)₂SO₄ (5.0), and MgSO₄ (0.5). Trace elements were as described by Rosenberger and Elsden (9). Cultures were incubated at 30°C for 48 h with rotary shaking at 180 rpm. Washed mycelia (typically 0.5 g [wet weight]) were resuspended in 40 ml of medium containing 10 mM morphine (Macfarlan Smith Ltd., Edinburgh, United Kingdom) in 250-ml Erlenmeyer flasks. Samples (0.2 ml) were removed at regular intervals and diluted fivefold in 50 mM phosphoric acid (pH 3.5), to dissolve any insoluble metabolites. Mycelia were removed by centrifugation at 14,000 × g with an MSE Microcentaur microcentrifuge (Patterson Scientific Ltd., Dunstable, United Kingdom). The samples were analyzed by high-performance liquid chromatography (HPLC) with a Waters component system (Millipore Waters UK Ltd., Watford, United Kingdom). The HPLC system consisted of a 600E system controller connected to either a 484 absorbance detector or a model 994 programmable photodiode array detector set to 230 nm, 0 to 1 V full-scale detection. Injections of 50 μl were performed with a WISP 712 autoinjector and data processed with Millennium 2010 software. Separation of samples was achieved with a C₁₈ Spherisorb column (4.6 by 250 mm, 5-μm particle size; Anachem Ltd., Luton, United Kingdom), protected by a guard column of the same packing material with an isocratic solvent system containing 40 mM phosphoric acid buffer (pH 2.5) and acetonitrile in a ratio of 92:8 plus 2 mM pentanesulfonic acid, delivered at a flow rate of 1 ml/min. Analysis of the whole-cell incubation mixture by HPLC showed that morphine was completely removed from the medium after a period of 70 h. No other soluble metabolites were identified by HPLC; however, a white precipitate was found to accumulate in the incubation mixture. Microscopic analysis showed that the precipitate had formed regular cubic crystals. The crystalline material was found to dissolve under mildly acidic conditions, and HPLC analysis after such treatment revealed the stoichiometric conversion of morphine to an unknown compound (Fig. ​(Fig.1)1) that had a retention time that coincided with that of authentic 2,2′-bimorphine, kindly provided by M. McPherson (Macfarlan Smith Ltd.). The compound was analyzed by thin-layer chromatography (TLC) with polyester-backed plates precoated with Polygram Sil G/UV₂₅₄ (Machery-Nagel, Duren, Germany) and developed in ammonia-n-butanol (20/80 [vol/vol]). TLC analysis revealed the appearance of two compounds that were detectable under UV light at 254 nm and with Ludy Tenger reagent (7). Compound 1 had an R_f value of 0.42 corresponding to that of authentic morphine, while compound 2 had an R_f value of 0.25 which coincided with that of authentic 2,2′-bimorphine. 2,2′-Bimorphine shows greatly enhanced fluorescence characteristics, compared to those of morphine, due to extended conjugation (6). Compound 2 fluoresced with a characteristic blue color when the TLC plate was illuminated at 366 nm. Fluorescent excitation and emission spectra of compound 2 dissolved in 50 mM potassium phosphate buffer (pH 7.4) in 1-cm-path-length cuvettes were recorded with a Perkin-Elmer LS 50 B luminescence spectrometer (Perkin-Elmer Ltd., Beaconsfield, United Kingdom). Two principal excitation maxima were found at 280 and 320 nm, with a single emission maximum at 430 nm, typical of authentic 2,2′-bimorphine. Open in a separate windowFIG. 1Accumulation of 2,2′-bimorphine in whole-cell incubations of C. didymum. Whole-cell incubations contained 40 ml of minimal medium, 10 mM morphine, and 0.5 g (wet weight) of mycelia in 250-ml Erlenmeyer flasks. Morphine (•) and 2,2′-bimorphine (○) concentrations were determined by HPLC. The data are means of three replicate incubations.

Identification of 2,2′-bimorphine.

¹H nuclear magnetic resonance spectroscopy of the product was performed at 400 MHz with a Bruker AM-400 spectrometer with tetramethylsilane as an internal standard and D-6 dimethyl sulfoxide as the solvent. The ¹H nuclear magnetic resonance spectrum gave the following signals, which were indistinguishable from those of an authentic sample of 2,2′-bimorphine (5) (for the proton assignments, see Fig. ​Fig.2,2, which gives the 2,2′-bimorphine numbering system): δ H 6.31 (2H, s, 1-H and 1′-H); 5.58 (2H, dd, J = 9.6 and 2.5, 7-H and 7′-H); 5.26 (2H, d, J = 9.6, 8-H and 8′-H); 4.70 (2H, d, J = 5.7, 5-H and 5′-H); 4.10 (2H, dd, J = 5.7 and 2.5, 6-H and 6′-H); 3.29 (2H, dd, J = 6.2 and 2.6, 9-H and 9′-H); 2.91 (2H, d, J = 18.6, 10β-H and 10β′-H); 2.57 (2H, d, J = 2.6, 14-H and 14′-H); 2.50 (2H, dd, J = 12.5 and 3.5, 16β-H and 16β′-H); 2.32 (6H, s, NMe and NMe′); 2.28 (2H, d, J = 12.5, α16-H and α16′-H); 2.23 (2H, dd, J = 18.6 and 6.2, α10-H and α10′-H); 1.99 (2H, dd, J = 11.4 and 3.5, α15-H and α15′-H); 1.68 (2H, d, J = 11.4, β15-H and β15′-H). Open in a separate windowFIG. 2Morphine analogs.The ¹H spectrum agreed with that expected for a symmetrical dimer, and only one aromatic proton signal was observed, instead of the characteristic AB pair of the morphine spectra, suggesting a symmetrical substitution on the aromatic ring. Laser desorption time-of-flight mass spectrometry was performed with a Kompact Maldi III mass spectrometer, and the mass spectrum showed a molecular ion, m/z 569.4, for C₃₄H₃₆N₂O₆.

Transformations of morphine analogs by C. didymum.

Whole-cell incubations of C. didymum were challenged with a range of morphine analogs including hydromorphone, 6-acetylmorphine, dihydromorphine, codeine, and diamorphine (see Fig. ​Fig.22 for structures). The incubations contained in 250-ml Erlenmeyer flasks approximately 0.61 g (wet weight) of mycelia and morphine analogs at 5 or 10 mM in a total volume of 40 ml of minimal medium. The flasks were incubated at 30°C with shaking, and samples were removed at intervals for HPLC analysis. Figure ​Figure33 shows that C. didymum was capable of activity with morphine, hydromorphone, 6-acetylmorphine, and dihydromorphine, and precipitates were observed to accumulate. Structural information on these products was not obtained. All of these compounds possess a free phenolic group at C-3 as a common structural feature which is likely to be an essential requirement for activity. This is consistent with the chemical oxidation of morphine to 2,2′-bimorphine, which requires the formation of a phenoxy radical intermediate (1). Open in a separate windowFIG. 3Transformations of morphine analogs by C. didymum. Whole-cell incubations contained 40 ml of minimal medium, 10 mM substrate (5 mM dihydromorphine), and 0.61 g (wet weight) of mycelia in 250-ml Erlenmeyer flasks. Morphine (•), codeine (▵), diamorphine (▴), hydromorphone (○), dihydromorphine (□), and 6-acetylmorphine (■) concentrations were determined by HPLC.

Enzyme activity in cell extracts.

The whole-cell transformation of morphine to 2,2′-bimorphine prompted investigation of subcellular enzyme activity. Cell extract was prepared by the method of Rahim and Sih (8) with the following modifications. Frozen mycelia containing 10 to 14 g (wet weight) of biomass were placed in an ice-cold mortar with an equal weight of acid-washed white quartz sand (50/70 mesh; Sigma Chemical Company, Poole, United Kingdom) and an equal volume of ice-cold potassium phosphate buffer (pH 7.4). The mixture was ground with a pestle for approximately 20 min until it formed a thin paste. The paste was diluted with an equivalent volume of ice-cold buffer, and the sand and cell debris were removed by centrifugation at 20,000 × g for 15 min at 4°C in a Sorvall RC5C centrifuge fitted with an SS34 rotor. Protein was measured by the method of Bradford (2) with the Pierce protein assay reagent according to the manufacturer’s protocol. Typically, protein recoveries of approximately 7 mg of protein/g (wet weight) of cells were obtained. The fluorescent nature of 2,2′-bimorphine enabled the development of a convenient and sensitive enzyme assay. In reaction mixtures which contained potassium phosphate buffer (pH 7.4), morphine (5 mM), and cell extract, activity could be measured spectrofluorimetrically by measuring fluorescence of 2,2′-bimorphine at 440 nm when excited at 330 nm. Cell extract from mycelia harvested after 80 h of incubation with morphine had a specific activity of 0.36 U/mg of protein. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of 2,2′-bimorphine from 2 μmol of morphine per min. No activity was observed in control reaction mixtures where the cell extract was replaced with boiled cell extract. Activity was inhibited completely when 0.1 mM azide was added to the reaction mixtures. Interestingly, no activity was observed in cell extract from mycelia that had not been incubated with morphine, which suggests that the activity is inducible. The development of a rapid and sensitive assay should facilitate the purification and characterization of the 2,2′-bimorphine-producing enzyme. 2,2′-Bimorphine has been shown to be a spontaneous reaction product of morphine in aqueous solutions, though the reaction was extremely slow (4). Furthermore, morphine can be oxidized to 2,2′-bimorphine with alkaline ferricyanide, a reaction which is known to proceed via a mesomeric aryloxy free radical, leading to the formation of the dimer (1). However, to the best of our knowledge, this is the first report of the microbial oxidation of morphine to 2,2′-bimorphine.     

Inhibition of anion transport in corn root protoplasts

The effects of several amino-reactive disulfonic stilbene derivatives and N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate on Cl⁻, SO₄²⁻, and inorganic phosphate (Pi) uptake in protoplasts isolated from corn root tissue were studied. 4-Acetamido-4′-isothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diamino-2,2′-stilbenedisulfonic acid, and NAP-taurine inhibited Cl⁻ and SO₄²⁻ but not Pi and K⁺ uptake in corn root protoplasts; whereas mersalyl inhibited Pi but not Cl⁻ or SO₄²⁻ uptake. The rate of uptake of all anions decreased with increasing external pH. In addition, these reagents markedly inhibited plasmalemma ATPase activity isolated from corn root tissue. Excised root segments were less sensitive to Cl⁻ and SO₄²⁻ transport inhibitors.     

Comparison of Gas Chromatography and Mineralization Experiments for Measuring Loss of Selected Polychlorinated Biphenyl Congeners in Cultures of White Rot Fungi

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl. The congener tested for mineralization was 2,4′,5-[U-¹⁴C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower, ≤11%, because it measures a complete oxidation of at least part of the congener molecule but the results were more consistent and therefore more reliable in assessment of PCB biodegradation.     

Characterization of the meta-Cleavage Compound Hydrolase Gene Involved in Degradation of the Lignin-Related Biphenyl Structure by Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 has the ability to transform a lignin-related biphenyl compound, 2,2′-dihydroxy-3,3′-dimethoxy-5,5′-dicarboxybiphenyl (DDVA), to 5-carboxyvanillic acid (5CVA) via 2,2′,3-trihydroxy-3′-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA). In the 4.9-kb HindIII fragment containing the OH-DDVA meta-cleavage dioxygenase gene (ligZ), we found a novel hydrolase gene (ligY) responsible for the conversion of the meta-cleavage compound of OH-DDVA to 5CVA. Incorporation of ¹⁸O from H₂¹⁸O into 5CVA indicated there was a hydrolytic conversion of the OH-DDVA meta-cleavage compound to 5CVA. LigY exhibited hydrolase activity only toward the meta-cleavage compound of OH-DDVA, suggesting its restricted substrate specificity.     

Characterization of Metabolites in the Biotransformation of 2,4,6-Trinitrotoluene with Anaerobic Sludge: Role of Triaminotoluene

The present study describes the biotransformation of 2,4,6-trinitrotoluene (TNT) (220 μM) by using anaerobic sludge (10%, vol/vol) supplemented with molasses (3.3 g/liter). Despite the disappearance of TNT in less than 15 h, roughly 0.1% of TNT was attributed to mineralization (¹⁴CO₂). A combination of solid-phase microextraction–gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry identified two distinctive cycles in the degradation of TNT. One cycle was responsible for the stepwise reduction of TNT to eventually produce triaminotoluene (TAT) in relatively high yield (160 μM). The other cycle involved TAT and was responsible for the production of azo derivatives, e.g., 2,2′,4,4′-tetraamino-6,6′-azotoluene (2,2′,4,4′-TA-6,6′-azoT) and 2,2′,6,6′-tetraamino-4,4′-azotoluene (2,2′,6,6′-TA-4,4′-azoT) at pH 7.2. These azo compounds were also detected when TAT was treated with the anaerobic sludge but not with an autoclaved sludge, suggesting the biotic nature of their formation. When the anaerobic conditions in the TAT-containing culture medium were removed by aeration and/or acidification (pH 3), the corresponding phenolic compounds, e.g., hydroxy-diaminotoluenes and dihydroxy-aminotoluenes, were observed at room temperature. Trihydroxytoluene was detected only after heating TAT in water at 100°C. When ¹³CH₃-labeled TNT was used as the N source in the above microcosms, we were unable to detect ¹³C-labeled p-cresol or [¹³CH₃]toluene, indicating the absence of denitration or deamination in the biodegradation process. The formation and disappearance of TAT were not accompanied by mineralization, suggesting that TAT acted as a dead-end metabolite.     

Evidence for an ATP-Dependent Proton Pump on the Golgi of Corn Coleoptiles

Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on sucrose density gradients, and ATP-dependent proton pumping activity was localized by the techniques of [¹⁴C]methylamine uptake and quinacrine fluorescence quenching. Two peaks of proton pumping activity were detected: a light peak (1.07 grams/cubic centimeter) corresponding to the previously characterized tonoplast-type H⁺-ATPase, and a second peak (1.13 grams/cubic centimeter) which coincided with the Golgi markers, latent UDPase, and glucan synthase I. The second peak was lighter than that of the plasma membrane marker, uridine diphosphoglucose-sterol glucosyltransferase (1.16 grams/cubic centimeter) and was not inhibited by vanadate, an inhibitor of the plasma membrane ATPase. The activity was also better correlated with the Golgi cisternae marker, glucan synthase I, than with latent UDPase, a secretory vesicle marker, but a secretory vesicle location cannot be ruled out. The tonoplast-type and Golgi proton pumps were similar in several respects, including a pH optimum at 7.2, stimulation by chloride, inhibition by diethylstilbestrol and N,N′-dicyclohexylcarbodiimide (DCCD), insensitivity to oligomycin and azide, and nucleotide specificity for Mg²⁺-ATP. However, the Golgi H⁺ pump was much less sensitive to nitrate and iodide, and more sensitive to the anion channel blockers, 4-acetamido-4′-isothiocyano-2,2′-stilbene sulfonic acid (SITS) and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) than the tonoplast-type H⁺-pump. The Golgi pump, but not the tonoplast-type pump, was stimulated by valinomycin in the presence of KCl. It is concluded that the Golgi of corn coleoptiles contains a KCl-stimulated H⁺-ATPase which can acidify the interior of Golgi cisternae and associated vesicles.     

Sequential Reductive Dechlorination of meta-Chlorinated Polychlorinated Biphenyl Congeners in Sediment Microcosms by Two Different Chloroflexi Phylotypes

Three species within a deeply branching cluster of the Chloroflexi are the only microorganisms currently known to anaerobically transform polychlorinated biphenyls (PCBs) by the mechanism of reductive dechlorination. A selective PCR primer set was designed that amplifies the 16S rRNA genes of a monophyletic group within the Chloroflexi including Dehalococcoides spp. and the o-17/DF-1 group. Assays for both qualitative and quantitative analyses by denaturing gradient gel electrophoresis and most probable number-PCR, respectively, were developed to assess sediment microcosm enrichments that reductively dechlorinated PCBs 101 (2,2′,4,5,5′-CB) and 132 (2,2′,3,3′,4,6′-CB). PCB 101 was reductively dechlorinated at the para-flanked meta position to PCB 49 (2,2′,4,5′-CB) by phylotype DEH10, which belongs to the Dehalococcoides group. This same species reductively dechlorinated the para- and ortho-flanked meta-chlorine of PCB 132 to PCB 91 (2,2′,3′,4,6′-CB). However, another phylotype designated SF1, which is more closely related to the o-17/DF-1 group, was responsible for the subsequent dechlorination of PCB 91 to PCB 51 (2,2′,4,6′-CB). Using the selective primer set, an increase in 16S rRNA gene copies was observed only with actively dechlorinating cultures, indicating that PCB-dechlorinating activities by both phylotype DEH10 and SF1 were linked to growth. The results suggest that individual species within the Chloroflexi exhibit a limited range of congener specificities and that a relatively diverse community of species within a deeply branching group of Chloroflexi with complementary congener specificities is likely required for the reductive dechlorination of different PCBs congeners in the environment.     

Aerobic Biodegradation of 2,2′-Dithiodibenzoic Acid Produced from Dibenzothiophene Metabolites

Dibenzothiophene is a sulfur heterocycle found in crude oils and coal. The biodegradation of dibenzothiophene through the Kodama pathway by Pseudomonas sp. strain BT1d leads to the formation of three disulfides: 2-oxo-2-(2-thiophenyl)ethanoic acid disulfide, 2-oxo-2-(2-thiophenyl)ethanoic acid-2-benzoic acid disulfide, and 2,2′-dithiodibenzoic acid. When provided as the carbon and sulfur source in liquid medium, 2,2′-dithiodibenzoic acid was degraded by soil enrichment cultures. Two bacterial isolates, designated strains RM1 and RM6, degraded 2,2′-dithiodibenzoic acid when combined in the medium. Isolate RM6 was found to have an absolute requirement for vitamin B₁₂, and it degraded 2,2′-dithiodibenzoic acid in pure culture when the medium was supplemented with this vitamin. Isolate RM6 also degraded 2,2′-dithiodibenzoic acid in medium containing sterilized supernatants from cultures of isolate RM1 grown on glucose or benzoate. Isolate RM6 was identified as a member of the genus Variovorax using the Biolog system and 16S rRNA gene analysis. Although the mechanism of disulfide metabolism could not be determined, benzoic acid was detected as a transient metabolite of 2,2′-dithiodibenzoic acid biodegradation by Variovorax sp. strain RM6. In pure culture, this isolate mineralized 2,2′-dithiodibenzoic acid, releasing 59% of the carbon as carbon dioxide and 88% of the sulfur as sulfate.     

Degradation of Polychlorinated Biphenyl Metabolites by Naphthalene-Catabolizing Enzymes

The ability of the dehydrogenase and ring cleavage dioxygenase of the naphthalene degradation pathway to transform 3,4-dihydroxylated biphenyl metabolites was investigated. 1,2-Dihydro-1,2-dihydroxynaphthalene dehydrogenase was expressed as a histidine-tagged protein. The purified enzyme transformed 2,3-dihydro-2,3-dihydroxybiphenyl, 3,4-dihydro-3,4-dihydroxybiphenyl, and 3,4-dihydro-3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl to 2,3-dihydroxybiphenyl, 3,4-dihydroxybiphenyl (3,4-DHB), and 3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl (3,4-DH-2,2′,5,5′-TCB), respectively. Our data also suggested that purified 1,2-dihydroxynaphthalene dioxygenase catalyzed the meta cleavage of 3,4-DHB in both the 2,3 and 4,5 positions. This enzyme cleaved 3,4-DH-2,2′,5,5′-TCB and 3,4-DHB at similar rates. These results demonstrate the utility of the naphthalene catabolic enzymes in expanding the ability of the bph pathway to degrade polychlorinated biphenyls.     

Characterization of the 2',3' cyclic phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase-phosphatase and bacteriophage lambda phosphatase

Clostridium thermocellum polynucleotide kinase-phosphatase (CthPnkp) catalyzes 5′ and 3′ end-healing reactions that prepare broken RNA termini for sealing by RNA ligase. The central phosphatase domain of CthPnkp belongs to the dinuclear metallophosphoesterase superfamily exemplified by bacteriophage λ phosphatase (λ-Pase). CthPnkp is a Ni²⁺/Mn²⁺-dependent phosphodiesterase-monoesterase, active on nucleotide and non-nucleotide substrates, that can be transformed toward narrower metal and substrate specificities via mutations of the active site. Here we characterize the Mn²⁺-dependent 2′,3′ cyclic nucleotide phosphodiesterase activity of CthPnkp, the reaction most relevant to RNA repair pathways. We find that CthPnkp prefers a 2′,3′ cyclic phosphate to a 3′,5′ cyclic phosphate. A single H189D mutation imposes strict specificity for a 2′,3′ cyclic phosphate, which is cleaved to form a single 2′-NMP product. Analysis of the cyclic phosphodiesterase activities of mutated CthPnkp enzymes illuminates the active site and the structural features that affect substrate affinity and k_cat. We also characterize a previously unrecognized phosphodiesterase activity of λ-Pase, which catalyzes hydrolysis of bis-p-nitrophenyl phosphate. λ-Pase also has cyclic phosphodiesterase activity with nucleoside 2′,3′ cyclic phosphates, which it hydrolyzes to yield a mixture of 2′-NMP and 3′-NMP products. We discuss our results in light of available structural and functional data for other phosphodiesterase members of the binuclear metallophosphoesterase family and draw inferences about how differences in active site composition influence catalytic repertoire.     

A new modified thrombin binding aptamer containing a 5'-5' inversion of polarity site

The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5′–5′ inversion of polarity site, namely d(^3′GGT^5′-^5′TGGTGTGGTTGG³′), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d(^3′GGT^5′-^5′TGGTGTGGTTGG^3′) is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4°C higher than the corresponding unmodified TBA. d(^3′GGT^5′-^5′TGGTGTGGTTGG^3′) continues to display an anticoagulant activity, even if decreased with respect to the TBA.     

Adenosine 5'-sulphatophosphate kinase activity in spinach leaf tissue

1. An F⁻-insensitive 3′-nucleotidase was purified from spinach leaf tissue; the enzyme hydrolysed 3′-AMP, 3′-CMP and adenosine 3′-phosphate 5′-sulphatophosphate but not adenosine 5′-nucleotides nor PP_i. The pH optimum of the enzyme was 7.5; K_m (3′-AMP) was approx. 0.8mm and K_m (3′-CMP) was approx. 3.3mm. 3′-Nucleotidase activity was not associated with chloroplasts. Purified Mg²⁺-dependent pyrophosphatase, free from F⁻-insensitive 3′-nucleotidase, catalysed some hydrolysis of 3′-AMP; this activity was F⁻-sensitive. 2. Adenosine 5′-sulphatophosphate kinase activity was demonstrated in crude spinach extracts supplied with 3′-AMP by the synthesis of the sulphate ester of 2-naphthol in the presence of purified phenol sulphotransferase; purified ATP sulphurylase and pyrophosphatase were also added to synthesize adenosine 5′-sulphatophosphate. Adenosine 5′-sulphatophosphate kinase activity was associated with chloroplasts and was released by sonication. 3. Isolated chloroplasts synthesized adenosine 3′-phosphate 5′-sulphatophosphate from sulphate and ATP in the presence of a 3′-nucleotide; the formation of adenosine 5′-sulphatophosphate was negligible. In the absence of a 3′-nucleotide the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate was negligible, but the formation of adenosine 5′-sulphatophosphate was readily detected. Some properties of the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate by isolated chloroplasts are described. 4. Adenosine 3′-phosphate 5′-sulphatophosphate, synthesized by isolated chloroplasts, was characterized by specific enzyme methods, electrophoresis and i.r. spectrophotometry. 5. Isolated chloroplasts catalysed the incorporation of sulphur from sulphate into cystine/cysteine; the incorporation was enhanced by 3′-AMP and l-serine. It was concluded that adenosine 3′-phosphate 5′-sulphatophosphate is an intermediate in the incorporation of sulphur from sulphate into cystine/cysteine.     

Evidence for Abasic Site Sugar Phosphate-Mediated Cytotoxicity in Alkylating Agent Treated Saccharomyces cerevisiae

To better understand alkylating agent-induced cytotoxicity and the base lesion DNA repair process in Saccharomyces cerevisiae, we replaced the RAD27^FEN1 open reading frame (ORF) with the ORF of the bifunctional human repair enzyme DNA polymerase (Pol) β. The aim was to probe the effect of removal of the incised abasic site 5′-sugar phosphate group (i.e., 5′-deoxyribose phosphate or 5′-dRP) in protection against methyl methanesulfonate (MMS)-induced cytotoxicity. In S. cerevisiae, Rad27^Fen1 was suggested to protect against MMS-induced cytotoxicity by excising multinucleotide flaps generated during repair. However, we proposed that the repair intermediate with a blocked 5′-end, i.e., 5′-dRP group, is the actual cytotoxic lesion. In providing a 5′-dRP group removal function mediated by dRP lyase activity of Pol β, the effects of the 5′-dRP group were separated from those of the multinucleotide flap itself. Human Pol β was expressed in S. cerevisiae, and this partially rescued the MMS hypersensitivity observed with rad27^fen1-null cells. To explore this rescue effect, altered forms of Pol β with site-directed eliminations of either the 5′-dRP lyase or polymerase activity were expressed in rad27^fen1-null cells. The 5′-dRP lyase, but not the polymerase activity, conferred the resistance to MMS. These results suggest that after MMS exposure, the 5′-dRP group in the repair intermediate is cytotoxic and that Rad27^Fen1 protection against MMS in wild-type cells is due to elimination of the 5′-dRP group.     

Characterization of in vitro proton pumping by microsomal vesicles isolated from corn coleoptiles

Corn (Zea mays L. cv Golden Cross Bantam) coleoptile microsomal vesicles have been isolated which are capable of ATP-driven H⁺-transport as measured by [¹⁴C]methylamine accumulation and quinacrine fluorescence quenching. Formation of the pH gradient in vitro shows a high specificity for ATP·Mg, is temperature-sensitive, exhibits a pH optimum at 7.5, and is inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Of the divalent cations tested, Mn²⁺ is almost as effective as Mg²⁺, while Ca²⁺ is ineffective. Excess divalent cations, particularly Ca²⁺, reduces the pH gradient. H⁺ transport is strongly promoted by anions, especially chloride, while potassium does not affect pump activity. Studies with ³⁶Cl⁻ indicate that ATP-driven H⁺ transport into the vesicles is associated with chloride uptake. Both carbonyl cyanide-m-chlorophenylhydrazone and the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, inhibit methylamine accumulation and ³⁶Cl⁻ uptake. Proton pumping is also blocked by diethyl stilbestrol and N,N′-dicyclohexylcarbodiimide, but is insensitive to oligomycin and vanadate. These properties of the pump are inconsistent with either a mitochondrial or plasma membrane origin.     

Regulation of Assimilatory Sulfate Reduction by Herbicide Safeners in Zea mays L

Effects of the herbicide safeners N,N-diallyl-2,2-dichloroacetamide and 4-dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzooxazin (CGA 154281) on the contents in cysteine and glutathione, on the assimilation of ³⁵SO₄²⁻, and on the enzymes of assimilatory sulfate reduction were analyzed in roots and primary leaves of maize (Zea mays) seedlings. Both safeners induced an increase in cysteine and glutathione. In labeling experiments using ³⁵SO₄²⁻, roots of plants cultivated in the presence of safeners contained an increased level of radioactivity in glutathione and cysteine as compared with controls. A significant increase in uptake of sulfate was only detected in the presence of CGA 154281. One millimolar N,N-diallyl-2,2-dichloroacetamide applied to the roots for 6 days increased the activity of adenosine 5′-phosphosulfate sulfotransferase about 20- and threefold in the roots and leaves, respectively, compared with controls. CGA 154281 at 10 micromolar caused a sevenfold increase of this enzyme activity in the roots, but did not affect it significantly in the leaves. A significant increase in ATP-sulfurylase (EC 2.7.7.4) activity was only detected in the roots cultivated in the presence of 10 micromolar CGA 154281. Both safeners had no effect on the activity of sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8). The herbicide metolachlor alone or combined with the safeners induced levels of adenosine 5′-phosphosulfate sulfotransferase, which were higher than those of the appropriate controls. Taken together these results show that the herbicide safeners increased both the level of adenosine 5′-phosphosulfate sulfotransferase activity and of the thiols cysteine and glutathione. This indicates that these safeners may be involved in eliminating the previously proposed regulatory mechanism, in which increased concentrations of thiols regulate assimilatory sulfate reduction by decreasing the activities of the enzymes involved.     

Structures of Bacterial Polynucleotide Kinase in a Michaelis Complex with Nucleoside Triphosphate (NTP)-Mg2+ and 5′-OH RNA and a Mixed Substrate-Product Complex with NTP-Mg2+ and a 5′-Phosphorylated Oligonucleotide

Clostridium thermocellum polynucleotide kinase (CthPnk), the 5′-end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from a nucleoside triphosphate (NTP) donor to a 5′-OH polynucleotide acceptor, either DNA or RNA. Here we report the 1.5-Å crystal structure of CthPnk-D38N in a Michaelis complex with GTP-Mg²⁺ and a 5′-OH RNA oligonucleotide. The RNA-binding mode of CthPnk is different from that of the metazoan RNA kinase Clp1. CthPnk makes hydrogen bonds to the ribose 2′-hydroxyls of the 5′ terminal nucleoside, via Gln51, and the penultimate nucleoside, via Gln83. The 5′-terminal nucleobase is sandwiched by Gln51 and Val129. Mutating Gln51 or Val129 to alanine reduced kinase specific activity 3-fold. Ser37 and Thr80 donate functionally redundant hydrogen bonds to the terminal phosphodiester; a S37A-T80A double mutation reduced kinase activity 50-fold. Crystallization of catalytically active CthPnk with GTP-Mg²⁺ and a 5′-OH DNA yielded a mixed substrate-product complex with GTP-Mg²⁺ and 5′-PO₄ DNA, wherein the product 5′ phosphate group is displaced by the NTP γ phosphate and the local architecture of the acceptor site is perturbed.     

Phosphate Translocator of Mesophyll and Bundle Sheath Chloroplasts of a C(4) Plant, Panicum miliaceum L. : Identification and Kinetic Characterization

The phosphate translocator was identified in the envelope membranes of both mesophyll and bundle sheath chloroplasts of Panicum miliaceum L. by labeling with [1,2-³H]1,2-(2,2′ -disulfo-4,4′ -diisothiocyano)diphenylethane ([³H]H₂DIDS) and by using SDS-PAGE. Assay of ³²Pi uptake by the chloroplasts showed that the phosphate translocators of both types of chloroplasts have a higher affinity for phosphoenolpyruvate than the C₃ counterpart and can be regarded as C₄ types.     

Identification, Cloning, and Characterization of a Novel Ketoreductase from the Cyanobacterium Synechococcus sp. Strain PCC 7942

A new ketoreductase useful for asymmetric synthesis of chiral alcohols was identified in the cyanobacterium Synechococcus sp. strain PCC 7942. Mass spectrometry of trypsin-digested peptides identified the protein as 3-ketoacyl-[acyl-carrier-protein] reductase (KR) (EC 1.1.1.100). The gene, referred to as fabG, was cloned, functionally expressed in Escherichia coli, and subsequently purified to homogeneity. The enzyme displayed a temperature optimum at 44°C and a broad pH optimum between pH 7 and pH 9. The NADPH-dependent KR was able to asymmetrically reduce a variety of prochiral ketones with good to excellent enantioselectivities (>99.8%). The KR showed particular high specific activity for asymmetric reduction of ethyl 4-chloroacetoacetate (38.29 ± 2.15 U mg⁻¹) and 2′,3′,4′,5′,6′-pentafluoroacetophenone (8.57 ± 0.49 U mg⁻¹) to the corresponding (S)-alcohols. In comparison with an established industrial enzyme like the alcohol dehydrogenase from Lactobacillus brevis, the KR showed seven-times-higher activity toward 2′,3′,4′,5′,6′-pentafluoroacetophenone, with a remarkably higher enantiomeric excess (>99.8% [S] versus 43.3% [S]).     

A candidate prostate cancer susceptibility gene encodes tRNA 3' processing endoribonuclease

tRNA 3′ processing endoribonuclease (3′ tRNase) is an enzyme responsible for the removal of a 3′ trailer from precursor tRNA (pre-tRNA). We purified ~85 kDa 3′ tRNase from pig liver and determined its partial sequences. BLAST search of them suggested that the enzyme was the product of a candidate human prostate cancer susceptibility gene, ELAC2, the biological function of which was totally unknown. We cloned a human ELAC2 cDNA and expressed the ELAC2 protein in Escherichia coli. The recombinant ELAC2 was able to cleave human pre-tRNA^Arg efficiently. The 3′ tRNase activity of the yeast ortholog YKR079C was also observed. The C-terminal half of human ELAC2 was able to remove a 3′ trailer from pre-tRNA^Arg, while the N‐terminal half failed to do so. In the human genome exists a gene, ELAC1, which seems to correspond to the C-terminal half of 3′ tRNase from ELAC2. We showed that human ELAC1 also has 3′-tRNase activity. Furthermore, we examined eight ELAC2 variants that seem to be associated with the occurrence of prostate cancer for 3′-tRNase activity. Seven ELAC2 variants which contain one to three amino acid substitutions showed efficient 3′-tRNase activities, while one truncated variant, which lacked a C-terminal half region, had no activity.