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Bernhard Kräutler

Publications and source records attributed to Bernhard Kräutler.

13 recordsLinked to original sources
PubMed2005

Chlorophyll breakdown in senescent Arabidopsis leaves. Characterization of chlorophyll catabolites and of chlorophyll catabolic enzymes involved in the degreening reaction.

During senescence, chlorophyll (chl) is metabolized to colorless nonfluorescent chl catabolites (NCCs). A central reaction of the breakdown pathway is the ring cleavage of pheophorbide (pheide) a to a primary fluorescent chl catabolite. Two enzymes catalyze this reaction, pheide a oxygenase (PAO) and red chl catabolite reductase. Five NCCs and three fluorescent chl catabolites (FCCs) accumulated during dark-induced chl breakdown in Arabidopsis (Arabidopsis thaliana). Three of these NCCs and one FCC (primary fluorescent chl catabolite-1) were identical to known catabolites from canola (Brassica napus). The presence in Arabidopsis of two modified FCCs supports the hypothesis that modifications, as present in NCCs, occur at the level of FCC. Chl degradation in Arabidopsis correlated with the accumulation of FCCs and NCCs, as well as with an increase in PAO activity. This increase was due to an up-regulation of Pao gene expression. In contrast, red chl catabolite reductase is not regulated during leaf development and senescence. A pao1 knockout mutant was identified and analyzed. The mutant showed an age- and light-dependent cell death phenotype on leaves and in flowers caused by the accumulation of photoreactive pheide a. In the dark, pao1 exhibited a stay-green phenotype. The key role of PAO in chl breakdown is discussed.

Apoptosis Regulatory Proteins↗
PubMed2005

Enhancing the methyl-donor activity of methylcobalamin by covalent attachment of DNA.

The preparation of a covalent DNA conjugate of vitamin B12 by means of heterogeneous solid-phase synthesis is reported. The cyano-corrinoid made available, dipotassium Co(beta)-cyanocobalamin-(3''-->2'),(3''-->5')-bis-2''-deoxythymidyl-3''-ate (K(2)-4), was cleanly methylated at the Co center by electrosynthetic means. Aqueous solutions of the resulting organometallic DNA-B12 conjugate K(2)-5 exhibited spectroscopic properties indicative of significant weakening of the axial (Co-N) bond, together with a 25-times higher basicity relative to Co(beta)-methylcobalamin (2). Methyl-transfer equilibria of pH-neutral aqueous solutions of K(2)-5 and cob(I)alamin (K-7) on one side, and of cob(I)alamin-(3''-->2'),(3''-->5')-bis-2''-deoxythymidyl-3''-ate (K(3)-8) and methylcobalamin (2) on the other, were studied at room temperature (Scheme 3). The NMR-derived data provided an equilibrium constant of ca. 0.3. Activation of K(2)-5 for abstraction of its Co-bound Me group by a nucleophile (such as cob(I)alamin) was, thus, indicated.

DNA↗
PubMed2005

Homoadenosylcobalamins as probes for exploring the active sites of coenzyme B12-dependent diol dehydratase and ethanolamine ammonia-lyase.

[Omega-(Adenosyl)alkyl]cobalamins (homoadenosylcobalamins) are useful analogues of adenosylcobalamin to get information about the distance between Co and C5', which is critical for Co-C bond activation. In order to use them as probes for exploring the active sites of enzymes, the coenzymic properties of homoadenosylcobalamins for diol dehydratase and ethanolamine ammonia-lyase were investigated. The kcat and kcat/Km values for adenosylmethylcobalamin were about 0.27% and 0.15% that for the regular coenzyme with diol dehydratase, respectively. The kcat/kinact value showed that the holoenzyme with this analogue becomes inactivated on average after about 3000 catalytic turnovers, indicating that the probability of inactivation during catalysis is almost 500 times higher than that for the regular holoenzyme. The kcat value for adenosylmethylcobalamin was about 0.13% that of the regular coenzyme for ethanolamine ammonia-lyase, as judged from the initial velocity, but the holoenzyme with this analogue underwent inactivation after on average about 50 catalytic turnovers. This probability of inactivation is 3800 times higher than that for the regular holoenzyme. When estimated from the spectra of reacting holoenzymes, the steady state concentration of cob(II)alamin intermediate from adenosylmethylcobalamin was very low with either diol dehydratase or ethanolamine ammonia-lyase, which is consistent with its extremely low coenzymic activity. In contrast, neither adenosylethylcobalamin nor adeninylpentylcobalamin served as active coenzyme for either enzyme and did not undergo Co-C bond cleavage upon binding to apoenzymes.

Binding Sites↗
PubMed2004

Homocoenzyme B12 and bishomocoenzyme B12: covalent structural mimics for homolyzed, enzyme-bound coenzyme B12.

Efficient electrochemical syntheses of "homocoenzyme B(12)" (2, Co(beta)-(5'-deoxy-5'-adenosyl-methyl)-cob(III)alamin) and "bishomocoenzyme B(12)" (3, Co(beta)-[2-(5'-deoxy-5'-adenosyl)-ethyl]-cob(III)alamin) are reported here. These syntheses have provided crystalline samples of 2 and 3 in 94 and 77 % yield, respectively. In addition, in-depth investigations of the structures of 2 and 3 in solution were carried out and a high-resolution crystal structure of 2 was obtained. The two homologues of coenzyme B(12) (2 and 3) are suggested to function as covalent structural mimics of the hypothetical enzyme-bound "activated" (that is, "stretched" or even homolytically cleaved) states of the B(12) cofactor. From crude molecular models, the crucial distances from the corrin-bound cobalt center to the C5' atom of the (homo)adenosine moieties in 2 and 3 were estimated to be about 3.0 and 4.4 A, respectively. These values are roughly the same as those found in the two "activated" forms of coenzyme B(12) in the crystal structure of glutamate mutase. Indeed, in the crystal structure of 2, the cobalt center was observed to be at a distance of 2.99 A from the C5' atom of the homoadenosine moiety and the latter was found to be present in the unusual syn conformation. In solution, the organometallic moieties of 2 and 3 were shown to be rather flexible and to be considerably more dynamic than the equivalent group in coenzyme B(12). The homoadenosine moiety of 2 was indicated to occur in both the syn and the anti conformations.

Bacterial Proteins↗
PubMed2004

Chlorophyll breakdown in tobacco: on the structure of two nonfluorescent chlorophyll catabolites.

In extracts of senescent leaves of the tobacco plant Nicotiana rustica, two colorless compounds with UV/VIS characteristics of nonfluorescent chlorophyll catabolites (NCCs) were detected and tentatively identified as Nr-NCCs. These two polar NCCs were found in similar amounts in the fresh extracts, and their constitutions could be determined by spectroscopic analysis. The data showed both of the two Nr-NCCs to have the same tetrapyrrolic core structure, as reported previously for all other NCCs from senescent higher plants. In the less polar catabolite, named Nr-NCC-2, this core structure was conjugated with a glucopyranose unit, as similarly discovered earlier in Bn-NCC-2, an NCC from oilseed rape (Brassica napus). The more polar NCC from tobacco leaves, Nr-NCC-1, carried an additional malonyl substituent at the 6'-OH group of the glucopyranosyl moiety. Partial (enzyme-catalyzed) hydrolysis of Nr-NCC-1 gave Nr-NCC-2, while enzyme-catalyzed malonylation of Nr-NCC-2 gave Nr-NCC-1, establishing the identity of their basic tetrapyrrole structure. In earlier work (on the polar NCCs from oilseed rape), only separate glucopyranosyl and malonyl functionalities were detected. Nr-NCC-1, thus, represents a further variant of the structures of NCCs from senescent higher plants and exhibits an unprecedented peripheral refunctionalization in chlorophyll catabolites.

Chlorophyll↗
PubMed2003

Axial solvent coordination in "base-fff" cob(II)alamin and related co(II)-corrinates revealed by 2D-EPR.

Detailed information on the structure of cobalt(II) corrinates is of interest in the context of studies on the coenzyme B(12) catalyzed enzymatic reactions, where cob(II)alamin has been identified as a reaction intermediate. Cob(II)ester (heptamethyl cobyrinate perchlorate) is found to be soluble in both polar and nonpolar solvents and is therefore very suitable to study solvent effects on Co(II) corrinates. In the literature, Co(II) corrinates in solution are often addressed as four-coordinated Co(II) corrins. However, using a combination of continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) and pulse ENDOR (electron nuclear double resonance) at different microwave frequencies we clearly prove axial ligation for Cob(II)ester and the base-off form of cob(II)alamin (B(12r)) in different solvents. This goal is achieved by the analysis of the g values, and the hyperfine couplings of cobalt, some corrin nitrogens and hydrogens, and solvent protons. These parameters are shown to be very sensitive to changes in the solvent ligation. Density functional computations (DFT) facilitate largely the interpretation of the EPR data. In the CW-EPR spectrum of Cob(II)ester in methanol, a second component appears below 100 K. Different cooling experiments suggest that this observation is related to the phase transition of methanol from the alpha-phase to the glassy state. A detailed analysis of the EPR parameters indicates that this transition induces a change from a five-coordinated (above 100 K) to a six-coordinated (below 100 K) Co(II) corrin. In a CH(3)OH:H(2)O mixture the phase-transition properties alter and only the five-coordinated form is detected for Cob(II)ester and for base-off B(12r) at all temperatures. Our study thus shows that the characteristics of the solvent can have a large influence on the structure of Co(II) corrinates and that comparison with the protein-embedded cofactor requires some caution. Finally, the spectral similarities between Cob(II)ester and base-off B(12r) prove the analogies in their electronic structure.

Cobalt↗
PubMed2002

Chlorophyll breakdown in spinach: on the structure of five nonfluorescent chlorophyll catabolites.

In extracts of senescent leaves of spinach (Spinacia oleracea), five colourless compounds with UV/Vis-characteristics of nonfluorescent chlorophyll catabolites (NCCs) were detected and tentatively named So-NCCs. The most abundant polar NCC in the leaves of this vegetable, So-NCC-2, had been isolated earlier and its constitution was determined by spectroscopic means. The catabolite So-NCC-2 was found to be an epimer of a polar NCC from barley (Hordeum vulgare), the first non-green chlorophyll catabolite from a higher plant to be structurally analyzed. Here, we report on the isolation of four additional So-NCCs from the extracts of senescent leaves of Sp. oleracea by two- (or multi-)stage chromatographic purification and on their structural characterization. The constitution of So-NCC-3 could be determined by spectroscopic analysis in combination with chemical correlation with a known NCC from Cercidiphyllum japonicum (Cj-NCC): So-NCC-3 was identified as the hydrolysis product of the methyl ester function of Cj-NCC. The less polar catabolite So-NCC-4 could be directly identified with Cj-NCC. Two further So-NCCs, So-NCC-1 and So-NCC-5, were detected only in trace amounts. Five structurally related nonfluorescent chlorophyll catabolites (So-NCCs) are thus present in senescent leaves of spinach. The structures of this set of So-NCCs indicate several peripheral refunctionalization reactions and inform on the late catabolic transformations during leaf senescence. The transformation of the tetrapyrrolic skeleton in chlorophyll catabolism in spinach and in C. japonicum is revealed to exhibit a common stereochemical pattern.

Journal Article↗