Cytokinin oxidase from auxin- and cytokinin-dependent callus cultures of tobacco (Nicotiana tabacum L.)

Cytokinin oxidase was extracted and partially purified from auxin- and cytokinin-dependent callus tissue of tobacco (Nicotiana tabacum L. cv. Wisconsin 38). The activity of the enzyme preparation was examined using an assay based on the conversion of tritiated N⁶-(²-isopentenyl)adenine ([2,8-³H]iP) to adenine. Cytokinin oxidase exhibited a temperature optimum at 45–50°C and a relatively high pH optimum (8.5–9.0). The apparent K_m value of the enzyme was 4.3 M for iP. On the basis of the substrate competition assays, iP was determined to be the preferred substrate of the enzyme. Substrate competition was also observed with zeatin and the cytokinin-active urea derivative Thidiazuron. Cytokinins bearing saturated isoprenoid side chains or cyclic side chain structures, as well as auxins and abscisic acid, had no effect on the conversion of [2,8-³H]iP. The cytokinin oxidase exhibited increased activity in the presence of copper-imidazole complex in the reaction mixture. Under optimal concentrations of copper (15 mM CuCl₂) and imidazole (100 mM), the enzyme activity was enhanced ca. 40-fold. Under these conditions the pH optimum was lowered to pH 6.0, whereas the temperature optimum, the apparent K_m value, and the substrate specificity were not altered. Most of the enzyme moiety did not bind to the lectin concanavalin A. The characteristics of cytokinin oxidase presented here suggest that a novel molecular form of the enzyme, previously identified and characterized in Phaseolus lunatus callus cultures (Kamínek and Armstrong (1990) Plant Physiol 93:1530), also occurs in cultured tobacco tissue.Abbreviations Ade adenine - iP N⁶-(²-isopentenyl)adenine - [2,8-³H]iP [2,8-³H]-N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - (diH)iP N⁶-isopentyladenine - (diH)Z dihydrozeatin - BAP N⁶-benzyladenine - ( _o OH)[9R]BAP N⁶-(o-hydroxybenzyl)adenosine - (mOH)[9R]BAP N⁶-(m-hydroxybenzyl)adenosine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA naphthalene-1-acetic acid - ABA abscisic acid - Con A concanavalin A     

Crystal structures of two- and three-dimensional polymeric complexes assembled by metal pseudohalides and 4-aminobenzoic acid via hydrogen bonds and covalent bonds

Four polymeric complexes [M(SCN)₂(4-abaH)₂]_n [M=Co(II) (1) or Cd(II) (2), 4-abaH=4-aminobenzoic acid], [Zn(N₃)(4-aba)]_n (3) and [Cd(N₃)(4-aba)(H₂O)]_n (4) were prepared from the reactions of 4-abaH with M(SCN)₂ [M=Co(II) or Cd(II)] and M(N₃)₂ [M=Zn(II) or Cd(II)] at different pH values. Their crystal structures have been determined by single-crystal X-ray diffraction. Both 1 and 2 consist of one-dimensional chains [M(μ-1,3-SCN)₂(4-abaH)₂]_n, in which each pair of the lateral carboxylic groups form double hydrogen bonds to furnish infinite two-dimensional sheets. In 3, the Zn(II) atoms are bridged by μ-1,1-azide groups and μ₂-carboxylate-O,O′ groups into an infinite zigzag chain featuring six-membered (ZnNZnOCO)_n rings, which are further connected by the 4-aba-N,O,O′ groups to generate a two-dimensional network. In 4, however, adjacent Cd(II) atoms are bridged by μ-1,1,3-azide groups to form an infinite chain with both four-membered Cd₂(μ-1,1-N₃)₂ and eight-membered Cd₂(μ-1,3-N₃)₂ rings. These chains are further connected by the 4-aba-N,O groups to generate a three-dimensional brickwall-like network. The results show significant effect of pH on the formation of the network structures.     

A survey of hydrogen bond geometries in the crystal structures of amino acids

An analysis of the geometries of the hydrogen bonds observed by neutron diffraction in thirt-two crystal structures of amino acids shows the following results. Of the 168 hydrogen bonds in the data set, 64 involve the zwitterion groups 

and $\text{C}$O₂. Another 18 are from

to sulphate or carbonyl oxygens. The majority, 46, of these

H … O bonds are three-centered (bifurcated). Nine are four-centered (trifurcated). The geometry in which the three-centered hydrogen bond involves both oxygens of the same carboxylate group is not especially favoured. When it does occur, one hydrogen bond is generally shorter and the other longer, than when the bonding involves oxygens on different carboxylate groups. The shortest hydrogen bonds are the OH … O $\text{C}$, from a carboxylic acid hydroxyl to a carboxylate oxygen, and NH … O$\text{C}$ when the nitrogen is the ring atom in histidine or proline. Carboxylate groups, on average, accept six hydrogen bonds, with no examples of less than four bonds. The reason for the large number of three-centered

H … O$\text{C}$ bonds is therefore a proton deficiency arising from the disparity between the tripled donor property of the

groups and the sextuple, on average, acceptor property of the carboxylate groups. There is good geometrical evidence for the existence of H … O and H … Cl^? hydrogen bonds, especially involving the hydrogen atoms on α-atoms.     

Ladder-like azido-bridged copper(II) complexes: Synthesis, X-ray structure and magnetic study

Two mixed-ligand copper(II) complexes [{Cu(L¹)(μ_1,3-N₃)}{Cu(L)(μ_1,3-N₃)(μ_1,1-N₃)}]_n (1) [HL¹ = 1-(N-ortho-hydroxyacetophenimino)-2,2-dimethyl-aminoethane; L = 2-(dimethylamino)-ethylamine] and [{Cu(L²)(μ_1,3-N₃)}{Cu(L)(μ_1,3-N₃)(μ_1,1-N₃)}]_n (2) [HL² = 1-(N-5-methoxy-ortho-hydroxyacetophenimino)-2,2-dimethyl-aminoethane] have been formed upon addition of aqueous solution of sodium azide to a methanolic solution of copper nitrate trihydrate and corresponding Schiff-base ligands. The ligands, HL¹ and HL² undergo partial hydrolysis of their imine bond during the course of reaction. Both the complexes contain single end-to-end (μ_1,3) azido bridged 1D infinite chains (rail) which propagate parallel to the crystallographic b-axis; neighboring chains are interconnected by pairs through double asymmetric end-on (μ_1,1) azido bridges (rung) to yield a ladder-like structure. In both complexes, rungs (end-on azido bridges) do not connect copper centers of the chains like in a regular ladder; instead they connect only the alternating copper sites of the 1D chain. In a chain the coordination environment around copper(II) ions are not the same: while the {Cu(L¹)(μ_1,3-N₃)} and {Cu(L²)(μ_1,3-N₃)} moieties have a penta-coordinated copper(II) center, the copper(II) ion of the neighboring {Cu(L¹)(μ_1,3-N₃)(μ_1,1-N₃)} or {Cu(L²)(μ_1,3-N₃)(μ_1,1-N₃)} moiety has an octahedral coordination environment. The variable temperature (2-300 K) magnetic susceptibility measurements showed that the magnetic interaction between the metal centers in complexes 1 and 2 is dominantly antiferromagnetic. The results of magnetic model are in good agreement with the experimental data.     

Effect of cytokinins on ribosomal RNA gene expression in excised cotyledons of Cucurbita pepo L.

Excised pumpkin (Cucurbita pepo L.) cotyledons were used to investigate the effects of two different types of cytokinins: N⁶-benzyladenine and N₁-(2-chloro-4-pyridyl)-N₂-phenylurea on RNA synthesis in isolated nuclei. Treatment of cotyledons with both cytokinins resulted in a rapid enhancement of nuclear RNA-polymerase-I activity (EC 2.7.7.6). Maximum stimulation of RNA polymerase I, responsible for rRNA synthesis, was observed 4–6 h after the start of cytokinin action. The activity of RNA polymerase II was stimulated much more slowly and to a lesser extent. Uridine 5-monophosphate-uridine analysis of the alkalidigested nascent pre-rRNA chains showed that the stimulation of RNA-polymerase-I activity was the consequence of an increase of the polyribonucleotide-clongation rate. No significant change in the number of transcribing enzyme molecules was defected after hormone treatment (86·10³ RNA-polymerase-I molecules per diploid genome).Indications that de-novo protein synthesis is necessary for cytokinin-mediated RNA-polymerase stimulation were derived from experiments showing inhibition by cycloheximide.Abbreviations BA N⁶-benzyladenine - [PU]-30 N₁-(2-chloro-4-pyridyl)-N₂-phenylurea - UMP undine 5-monophosphate - UTP udine 5-triphosphate     

Isolation of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus.

The structure of α-chitin has been determined by X-ray diffraction, based on the intensity data from deproteinized lobster tendon. Least-squares refinement shows that adjacent chains have alternating sense (i.e. are antiparallel). In addition, there is a statistical distribution of side-chain orientations, such that all the hydroxyl groups form hydrogen bonds. The unit cell is orthorhombic with dimensions a = 0.474 ± 0.001 nm, b = 1.886 ± 0.002 nm and c = 1.032 ± 0.002 nm (fiber axis); the space group is P2₁2₁2₁ and the cell contains disaccharide sections of the two chains passing through the center and corner of the ab projection. The chains form hydrogen-bonded sheets linked by CO…HN bonds approximately parallel to the a axis, and each chain has an O-3′H…O.5 intramolecular hydrogen bond, similar to that in cellulose. Adjacent chains along the ab diagonal have different conformations for the CH₂OH groups: on one chain these groups form O.6H…O.6′ intermolecular hydrogen bonds to the CH₂OH group on the adjacent chain along the ab diagonal. The latter group is oriented to form an intramolecular O.6′H…O.7 bond to the carboxyl oxygen on the next residue. The results indicate that a statistical mixture of CH₂OH orientations is present, equivalent to half oxygens on each residue, each forming inter- and intramolecular hydrogen bonds. As a result the structure contains two types of amide groups, which differ in their hydrogen bonding, and account for the splitting of the amide I band in the infrared spectrum. The Inability of this chitin polymorph to swell on soaking in water is explained by the extensive intermolecular hydrogen bonding.     

Theoretical study on the binding mechanism between N6-methyladenine and natural DNA bases

N6-methyladenine (m⁶A) is a rare base naturally occurring in DNA. It is different from the base adenine due to its N-CH₃. Therefore, the base not only pairs with thymine, but also with other DNA bases (cytosine, adenine and guanine). In this work, Møller-Plesset second-order (MP2) method has been used to investigate the binding mechanism between m⁶A and natural DNA bases in gas phase and in aqueous solution. The results show that N-CH₃ changed the way of N6-methyladenine binding to natural DNA bases. The binding style significantly influences the stability of base pairs. The trans-m⁶A:G and trans-m⁶A:C conformers are the most stable among all the base pairs. The existence of solvent can remarkably reduce the stability of the base pairs, and the DNA bases prefer pairing with trans-m⁶A to cis-m⁶A. Besides, the properties of these hydrogen bonds have been analyzed by atom in molecules (AIM) theory, natural bond orbital (NBO) analysis and Wiberg bond indexes (WBI). In addition, pairing with m⁶A decreases the binding energies compared to the normal Watson-Crick base pairs, it may explain the instability of the N6 site methylated DNA in theory.

Fractionation and specificity of DNA methylases from the Escherichia coli SK cells

Summary Specificity of DNA methylation enzymes from the E. coli SK cells and conditions for their separation have been investigated. Column chromatography on carboxymethylcellulose permits fractionation of methylase activity into six discrete peaks whose specificity to the methylated base has been determined in vitro with H³-SAM as precursor. All methylases specific for adenine produced 6-methylaminopurine, all methylases specific for cytosine yielded 5-methylcytosine.The first enzymatic activity peak containing cytosine methylase free of traces of adenine-methyiating activity (E₁), and the second peak containing both the enzymes (E₂) were not adsorbed on the ion exchanger and went off the column with the effluent (column buffer). Adenine specific methylase E₂ is retarded to a small extent during the passage through the column. The second adenine methylases (W) was characterized by weak bonds with the ion exchanger and was removed when washing the column with column buffer. The elution with NaCl gradient produced successively three enzymatic activity peaks: adenine methylase (G_I), cytosine methylase (G_II), and one more adenine methylase (G_III) removed from the column by 0.16 m, 0.24 m and 0.43 m NaCl respectively.Using a new modification of the complementary methylation test, the specificity with regard to recognition site was examined for all enzymes, except for W and G_III, which were extremely unstable. The adenine methylases E₂ and G_I and the cytosine methylases E₁ and G_II were shown to recognize different sites and to be different enzymes. In view of the drastic differences in their chromatographic behaviour and physical stability, the adenine methylases W and G_III are probably also different enzymes.     

Theoretical investigation on the water-assisted excited-state proton transfer of 7-azaindole derivatives: substituent effect

A systematic study on the first excited-state proton transfer (ESPT) in 2RAI-H₂O (R = OH, OCH₃, CN, NO₂, CHO) complexes in solution were investigated at the TD-M06-2X/6-31 + G(d, p) level. The double proton transfer occurred in an asynchronous but concerted protolysis fashion no matter which substituent R was used at C₂ position in pyrrole ring in the 7AI-H₂O complex. The specific vibrational-mode of ESPT in the model systems was confirmed and contributed to promote the reaction rate by shortening the reaction path. The substituent effects of different groups on the ESPT thermodynamics and kinetics were discussed. It was obvious that the geometries, barrier height, asynchrony, and specific vibration-mode of excited-state proton transfer changed with the different substituent groups.

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Graphical Abstract The distance between two neighboring heavy atoms such as N₁-O₁₁ (R₁) and O₁₁-N₈ (R₂) distances played an important role in the proton transfer reaction. The sum of the N₁-O₁₁ and O₁₁-N₈ distances in the reactant of 2RAI-H₂O (R=H, OH, OCH₃; CN, CHO, NO₂) complexes is in the range of 5.542 Å~5.692 Å and changes along with the substituent group at C₂ position in the pyrrole ring. The ESDPT barrier height and the sum of the N₁-O₁₁ and O₁₁-N₈ distances have a good correlation.

     

Thermodynamics and nuclear magnetic resonance studies of lanthanide complexation by ethylenediamine-NN′-diacetate-NN′-di-3-propionate

The proton NMR spectra of ethylenediamine-NN′- diacetate-NN′-di-3-propionate and its complexes with alkaline earth and diamagnetic lanthanide ions are described. Quartet splittings of the methylenic protons of the acetate groups upon complexation with metal ions of high charge density is indicative of long-lived metalnitrogen bonds and short-lived metaloxygen bonds. The observation of two AB quartets for the acetate protons complexed to trivalent ions was attributed to an unsymmetrical distribution of the acetate arms around the central ion. For Mg²⁺ complex, the two acetate arms are equivalently disposed and the two quartets collapse into one. For the other alkaline earths, a singlet is observed, indicating that the metalnitrogen bond lifetime is also short. The spectra of the propionate protons are consistent of either an ABCD (LuENDPDA and YENDPDA) or an AA′BB′ (MgENDPDA) spectral pattern. The thermodynamic data support these conclusions and show that substitution of C₂H₄CO₂⁻ groups for CH₂CO₂⁻ groups decreases the stability of the complex. This results from weakening of the metalnitrogen interaction due to the expansion of the

     

The influence of Cu+ binding to hypoxanthine on stabilization of mismatches involving hypoxanthine and DNA bases: a DFT study

In the present work, the influence of Cu⁺ binding to N3- and N7-positions of hypoxanthine on energetic, geometrical and topological properties of hypoxanthine–guanine, hypoxanthine–adenine, hypoxanthine–cytosine, hypoxanthine–thymine and hypoxanthine–hypoxanthine mismatches is theoretically investigated. The calculations, in gas phase, are performed at B3LYP/6-311++G(3df,3pd) level of theory. Unlike the other mispairs, Cu⁺ binding to N3-position of hypoxanthine causes the proton transfer process from enol form of hypoxanthine to imino forms of adenine and cytosine. This process also occurs in all mismatches having enol form of hypoxanthine when Cu⁺ binds to N7-position of hypoxanthine. The mismatches are stabilized by hydrogen bonds. The influence of Cu⁺ on hydrogen bonds is also examined by atoms in molecules (AIM) and natural bond orbital (NBO) analyses.

Communicated by Ramaswamy H. Sarma     

基于密度泛函理论研究Watson–Crick碱基对中的氢键特征(英文)

基于密度泛函理论(DFT)研究腺嘌呤、胸腺嘧啶、鸟嘌呤、胞嘧啶以及腺嘌呤胸腺嘧啶碱基对、鸟嘌呤胞嘧啶碱基对。在DFT-B3LYP/6-31G**水平上利用自然键轨道理论分析研究结果显示,互补碱基对的结构和电子特征有利于氢键的形成。本文中讨论几何结构、电子结构、分子轨道和能量对于氢键形成的影响。此研究结果将有助于更好的理解AT和GC碱基对中氢键与它们的结构特性之间的关系。     

Photoaffinity labeling of ribulose-bisphosphate carboxylase/oxygenase with 8-azidoadenosine 5′-triphosphate

Photoaffinity labeling with [³²P] 8-azidoadenosine 5-triphosphate (8-N₃ATP) was used to identify putative binding sites on tobacco (Nicotiana tabacum L. and N. rustica L.) leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase, EC 4.1.1.39). Incorporation of ³²P was observed in polypeptides corresponding to both RuBPCase subunits when desalted leaf and chloroplast extracts, and purified RuBPCase were irradiated with ultraviolet light in the presence of [³²P] 8-N₃ATP. ³²P-labeling was dependent upon ultraviolet irradiation and occurred with [³²P] 8-N₃ATP labeled in the -position, indicating covalent incorporation of the photoprobe. Both [³²P] 8-N₃ATP and [³²P] 8-N₃GTP were incorporated to a similar extent into the 53-kilodalton (kDa) large subunit (LSu), but incorporation of [³²P] 8-N₃GTP into the 14-kDa small subunit (SSu) of RuBPCase was <5% of that measured with [³²P] 8-N₃ATP. Distinct binding sites for 8-N₃ATP on the two subunits were indicated by different apparent K _D values, 3 and 18 M for the SSu and LSu, respectively, and differences in the response of photoaffinity labeling to Mg²⁺, anions and enzyme activation. Active-site-directed compounds, including the non-gaseous substrate ribulose 1,5-bisphosphate, the reaction intermediate analog 2-carboxyarabinitol-1,5-bisphosphate and several phosphorylated effectors afforded protection to the LSu site against photoincorporation but provided almost no protection to the SSu. These results indicate that 8-N₃ATP binds to the active-site region of the LSu and a distinct site on the SSu of RuBPCase. Experiments conducted with intact pea (Pisum sativum L.) and tobacco chloroplasts showed that the SSu was not photolabeled with [³²P] 8-N₃ATP in organello or in undesalted chloroplast lysates but was photolabeled when lysates were ultrafiltered or desalted. These results indicate that 8-N₃ATP binds to a site on the SSu that has physiological significance.Abbreviations kDa kilodalton - LSu large subunit - 8-N₃ATP 8-azidoadenosine 5-triphosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SSu small subunit Kentucky Agricultural Experiment Station Journal Article No. 89-3-150The authors acknowledge the technical assistance of J.C. Anderson. This work was supported in part by National Institute of Health grant GM 35766 to B.E.H.     

Interchain hydrogen bonds via bound water molecules in the collagen triple helix

If the collagen triple helix is so built as to have one set of NH ? O hydrogen bonds of the type N₃H₃(A) ? O₂(B), then it is possible to have a linkage between N₁H₁(B) and O₁(A) through the intermediary of a water molecule with an oxygen O leading to the formation of the hydrogen bonds N₁(B) ? O and O (A). In the same configuration, another water molecule with an oxygen O can link two earbonyl oxygens of chains A and B forming the hydrogen bonds O O₁(A) and O O₀ (B). The two water oxygens also become receptors at the same time for CH ? O hydrogen bonds. Thus, the neighboring chains in the triple helix are held together by secondary valence bond linkages occurring regularly sit intervals of about 3 Å along the length of the protofibril. The additional water molecules occur on the periphery of the proto-fibril and will contribute their full share towards stabilizing the structure in the solid state. In solution, they will be disturbed by the medium unless they are protected by long side groups. It appears that this type of two-bonded structure, in which one NH ? O bond is to a water molecule, can explain several observations on the stability and hydrogen exchange properties of collagen itself and related synthetic polypeptides. The nature of the water bonds and their strength are found to be better in the one-bonded structure proposed from Madras than in the one having the coordinates of Rich and Crick.     

Synthesis and x-ray structure of a Hg(II) complex with adenine N(1)-oxide. A model for mercury binding to DNA

The synthesis and crystal structure of the adenine N(1)-oxide complex with mercury(II) chloride, (C₅H₅N₅O)HgCl₂ are reported. Crystals of the coordination compound belong to the monoclinic system, space group P2₁/n with the following primary crystallographic data: a = 6.685(1) Å, b = 11.798(2) Å, c = 10.155(1) Å, β = 100.22(1)°, V = 906.04 ų, Z = 4. The structure was elucidated by conventional Patterson and Fourier methods and refined by the full matrix least-squares technique on the basis of 1977 observed reflections to an R value of 0.074. The basic unit of the structure is a dimer, with a centre of symmetry, consisting of two HgCl₂ moieties and two adenine N(1)-oxide ligands. A polymeric structure results from the bridging interactions of chloride ions. Adenine N(1)-oxide acts as a bidentate bridging ligand, coordinating through N(7) and O(1). The coordination geometry around the mercury ion is a distorted square pyramid with N(7) and three chlorines (two of which are centro-symmetrically related) forming the square plane and O(1) occupying the axial position. Hg also interacts indirectly with N(6) through a Cl

HN hydrogen bond. Principal intracomplex geometrical parameters are as follows: HgN(7) = 2.61(1) Å, HgO(1) = 2.55(1) Å, HgCl(1) = 2.330(3) Å, HgCl(2) = 2.318(3) Å, HgCl(2′) = 3.347(3) Å. The cis angles range from 77.5° to 107.9° and the two trans angles are 155.5° and 163.1°. The centro-symmetrically related bases overlap partially and pack at a distance of 3.2 Å. The glide-related bases are linked by a hydrogen bond, N(9)H

O(1) and are inclined to one another by 109.7°. The results are compared with those derived from spectroscopic and other physicochemical studies on metal interaction with adenine N(1)-oxide. Based on the present structural observations and earlier experimental results a possible mechanism is proposed for mercury interaction with DNA.     

A two-stranded template-based approach to G.(C-A) triad formation: designing novel structural elements into an existing DNA framework

We have designed a DNA sequence, d(G-G-G-T-T-C-A-G-G), which dimerizes to form a 2-fold symmetric G-quadruplex in which G(syn). G(anti).G(syn).G(anti) tetrads are sandwiched between all trans G. (C-A) triads. The NMR-based solution structural analysis was greatly aided by monitoring hydrogen bond alignments across N-H...N and N-H...O==C hydrogen bonds within the triad and tetrad, in a uniformly ((13)C,(15)N)-labeled sample of the d(G-G-G-T-T-C-A-G-G) sequence. The solution structure establishes that the guanine base-pairs with the cytosine through Watson-Crick G.C pair formation and with adenine through sheared G.A mismatch formation within the G.(C-A) triad. A model of triad DNA was constructed that contains the experimentally determined G.(C-A) triad alignment as the repeating stacked unit.