Methylated flavonoids from cistus ladanifer and cistuspalhinhae and their taxonomic implications

Three methylated kaempferol and two methylated apigenin derivatives were identified in the leaf resins of Cistus ladanifer and C. palhinhae. The two species produced identical secreted flavonoids which supports their close affinity based on morphological similarities.     

Synthesis, structure and bonding of cadmium(II) thiocyanate systems featuring nitrogen based ligands of different denticity

Complexes catena-[di(4-amino-pyridine)di(μ-S,N-thiocyanato)cadmium(II)], , catena-[{(1-pyridine-2-yl-ethylene)-hydrazine}di(μ-S,N-thiocyanato)cadmium(II)], , and di-μ-S,N-thiocyanatobis{(N,N-diethyl-N′-(1-pyridine-2-yl-ethylidene)-ethane-1,2-diamine)(N-thiocyanato)cadmium(II)}, [Cd(NCS)(μ-SCN)(L³)]₂ (3) have been synthesized by reacting cadmium acetate/NH₄SCN with 4-amino-pyridine (L¹), C₅H₄N-C(CH₃)NNH₂ (L²), and C₅H₄N-C(CH₃)N-CH₂-CH₂-N(C₂H₅)₂ (L³), respectively, in methanol. Characterization by single-crystal X-ray crystallography shows that in compounds 1 and 2 the cadmium atoms have a 4N2S-hexa-coordination sphere, exhibiting pseudo-octahedral geometry. The cadmium atoms are bridged by two thiocyanate ions generating 1-D polymeric chains. Compound 3 is a centrosymmetric dimeric complex, with the cadmium atom pseudo octahedrally surrounded by a 5N1S coordination sphere. In compound 1 the crystal packing is controlled mainly by interchain N-H?N and C-H?π interactions between the aminopyridine moieties, whereas in complexes 2 and 3 π-stacking interactions between the pyridyl planes stabilize the interchain or intermolecular packing, respectively. Thiocyanate and pyridylimine chelation to metal center is also scrutinized with EHMO analysis.     

Sequencing and heterologous expression of an epimerase and two lyases from iminodisuccinate-degrading bacteria

Recently, degradation of all existing epimers of the complexing agent iminodisuccinate (IDS) in the bacterial strain Agrobacterium tumefaciens BY6 was proven to depend on an epimerase and a C-N lyase (Cokesa et al., Appl. Environ. Microbiol. 70:3941-3947, 2004). In the bacterial strain Ralstonia sp. strain SLRS7, a corresponding C-N lyase is responsible for the initial degradation step (Cokesa et al., Biodegradation 15:229-239, 2004). The ite gene, encoding the IDS-transforming epimerase, and the genes icl(B) and icl(S), encoding the IDS-converting BY6-lyase and SLRS7-lyase, respectively, were cloned and sequenced. The epimerase gene encodes a protein with a predicted subunit molecular mass of 47.6 kDa. The highest degree of epimerase amino acid sequence identities was found with proteins of unknown function, indicating a novel protein. For the lyases, the deduced amino acid sequences show high similarity to enzymes of the fumarase II family. A classification into a new subfamily within the enzyme family is proposed. The subunit molecular masses of the lyases were calculated to be 54.4 and 54.7 kDa, respectively. In Agrobacterium tumefaciens BY6, the ite gene was on an approximately 180-kb circular plasmid, whereas the icl(B) gene was chromosomal like the corresponding icl(S) gene in Ralstonia sp. strain SLRS7. Heterologous expression in Escherichia coli and subsequent purification revealed recombinant enzymes with in vitro activity similar to that of the corresponding enzymes from the wild-type strains.     

Three-dimensional structure of iminodisuccinate epimerase defines the fold of the MmgE/PrpD protein family

Iminodisuccinate (IDS) epimerase catalyzes the epimerisation of R,R-, S,S- and R,S- iminodisuccinate, one step in the biodegradation of the chelating agent iminodisuccinate by Agrobacterium tumefaciens BY6. The enzyme is a member of the MmgE/PrpD protein family, a diverse and little characterized class of proteins of prokaryotic and eukaryotic origin. IDS epimerase does not show significant overall amino acid sequence similarity to any other protein of known three-dimensional structure. The crystal structure of this novel epimerase has been determined by multi-wavelength diffraction to 1.5 A resolution using selenomethionine-substituted enzyme. In the crystal, the enzyme forms a homo-dimer, and the subunit consists of two domains. The larger domain, not consecutive in sequence and comprising residues Met1-Lys266 and Leu400-Pro446, forms a novel all alpha-helical fold with a central six-helical bundle. The second, smaller domain folds into an alpha+beta domain, related in topology to chorismate mutase by a circular permutation. IDS epimerase is thus not related in three-dimensional structure to other known epimerases. The fold of the IDS epimerase is representative for the whole MmgE/PrpD family. The putative active site is located at the interface between the two domains of the subunit, and is characterized by a positively charged surface, consistent with the binding of a highly negatively charged substrate such as iminodisuccinate. Docking experiments suggest a two-base mechanism for the epimerisation reaction.     

A Stereoselective Carbon-Nitrogen Lyase from Ralstonia sp. SLRS7 Cleaves Two of Three Isomers of Iminodisuccinate

Following biodegradation tests according to the OECD guidelines for testing of chemicals 301F different degradation rates were observed for the three stereoisomers of iminodisuccinate (IDS). A strain was isolated from activated sludge, which used two of three isomers, R,S-IDS and S,S-IDS, as sole source of carbon, nitrogen, and energy. The isolated strain was identified by 16S-rDNA and referred to as Ralstonia sp. SLRS7. An IDS-degrading lyase was isolated from the cell-free extract. The enzyme was purified by three chromatographic steps, which included anion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. The lyase catalysed the non-hydrolytic cleavage of IDS without requirement of any cofactors. Cleavage of S,S-IDS led to the formation of fumaric acid and L-aspartic acid. Interestingly R,S-IDS yielded only D-aspartic acid besides fumaric acid. R,R-IDS was not transformed. Thus, the IDS-degrading enzyme is a carbon-nitrogen lyase attacking only the asymmetric carbon atom exhibiting the S-configuration. Besides S,S-IDS and R,S-IDS cleavage, the lyase catalysed also the transformation of certain S,S-IDS metal complexes, namely Ca(2+)-, Mg(2+)- and Mn(2+)-IDS. The maximum enzyme activity was found at pH 8.0-8.5 and 35 degrees C. SDS-PAGE analysis revealed a single 57-kDa protein band. The native enzyme was estimated to be around 240 kDa indicating a homotetramer enzyme.     

Hydride-Meisenheimer Complex Formation and Protonation as Key Reactions of 2,4,6-Trinitrophenol Biodegradation by Rhodococcus erythropolis

Biodegradation of 2,4,6-trinitrophenol (picric acid) by Rhodococcus erythropolis HLPM-1 proceeds via initial hydrogenation of the aromatic ring system. Here we present evidence for the formation of a hydride-Meisenheimer complex (anionic ς-complex) of picric acid and its protonated form under physiological conditions. These complexes are key intermediates of denitration and productive microbial degradation of picric acid. For comparative spectroscopic identification of the hydride complex, it was necessary to synthesize this complex for the first time. Spectroscopic data revealed the initial addition of a hydride ion at position 3 of picric acid. This hydride complex readily picks up a proton at position 2, thus forming a reactive species for the elimination of nitrite. Cell extracts of R. erythropolis HLPM-1 transform the chemically synthesized hydride complex into 2,4-dinitrophenol. Picric acid is used as the sole carbon, nitrogen, and energy source by R. erythropolis HLPM-1.     

Kinetic Study of Neuropeptide Y (NPY) Proteolysis in Blood and Identification of NPY3�C35: A NEW PEPTIDE GENERATED BY PLASMA KALLIKREIN*

There is little information on how neuropeptide Y (NPY) proteolysis by peptidases occurs in serum, in part because reliable techniques are lacking to distinguish different NPY immunoreactive forms and also because the factors affecting the expression of these enzymes have been poorly studied. In the present study, LC-MS/MS was used to identify and quantify NPY fragments resulting from peptidolytic cleavage of NPY_1–36 upon incubation with human serum. Kinetic studies indicated that NPY_1–36 is rapidly cleaved in serum into 3 main fragments with the following order of efficacy: NPY_3–36 ≫ NPY_3–35 > NPY_2–36. Trace amounts of additional NPY forms were identified by accurate mass spectrometry. Specific inhibitors of dipeptidyl peptidase IV, kallikrein, and aminopeptidase P prevented the production of NPY_3–36, NPY_3–35, and NPY_2–36, respectively. Plasma kallikrein at physiological concentrations converted NPY_3–36 into NPY_3–35. Receptor binding assays revealed that NPY_3–35 is unable to bind to NPY Y1, Y2, and Y5 receptors; thus NPY_3–35 may represent the major metabolic clearance product of the Y2/Y5 agonist, NPY_3–36.Neuropeptide Y (NPY)² is a 36-amino acid peptide involved in the central and peripheral control of blood pressure (1–4) and in feeding behavior and obesity (5–9). NPY stimulates at least 6 types of receptors, called Y1, Y2, Y3, Y4, Y5, and y6 (10–12). The Y1 receptor has high affinity for full-length NPY, while Y2 and Y5 receptors bind and are stimulated by full-length and N-terminally truncated NPY. The physiological effects associated to the Y1 and Y2 receptors are the best known; exposure to a Y1 agonist causes an increase in blood pressure and potentiates postsynaptically the action of other vasoactive substances (1, 4, 13), whereas Y2 receptors are mainly located presynaptically, and upon stimulation mediate the inhibition of neurotransmitter release (14, 15). NPY is a prototype of peptide whose function can be altered by proteases. Among peptidases displaying a high affinity for NPY, the primary role appears to be played by dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), a serine-type protease, also known as CD26, that releases an N-terminal dipeptide, Xaa-Xab- -Xac, preferentially when Xab is a proline or an alanine residue (16). By cleaving the Tyr-Pro dipeptide off the NPY N-terminal extremity, DPPIV generates NPY_3–36, a truncated form that loses its affinity for the Y1 receptor and becomes a Y2/Y5 receptor agonist (17, 18).NPY can also be degraded by aminopeptidase P (AmP, EC 3.4.11.9), a metalloprotease that hydrolyzes the peptide bond between the first and the second amino acid residue at the N terminus of proteins, if the second amino acid is a proline (19). AmP removes the N-terminal tyrosine from NPY to generate NPY_2–36, a selective Y2 agonist (18, 20). There is little information on how NPY cleavage by these enzymes occurs in serum, in part because reliable techniques are lacking to distinguish different NPY immunoreactive (NPYir) forms and also because the factors affecting the expression of these enzymes have been poorly studied. Recently, Frerker et al. (21) reported by MALDI-TOF mass spectrometry that NPY_1–36 is exclusively degraded by DPPIV into NPY_3–36 in EDTA-plasma but they did not provide kinetics of NPY cleavage efficiency of DPPIV. Beck-Sickinger and co-workers (22) studied with the same technique the metabolic stability of fluorescent N-terminally labeled NPY analogues incubated in human plasma and found that the 36th, 35th, and 33rd residues of NPY analogues may also be removed by unknown carboxypeptidases.We have set up a method using liquid chromatography coupled with tandem mass spectrometry (LC-MSⁿ) to selectively quantify NPY and its C-terminal fragments NPY_2–36 and NPY_3–36 digested by human serum. The assays used the internal standard methodology with stable isotopes NPY_1–36 (IDA) (23, 24) or porcine NPY_1–36 as internal standard.The goal of this work was: 1) to determine to which extent NPY_1–36 is degraded by proteases present in human serum and whether an inhibition of DPPIV and AmP by vildagliptin and apstatin (two specific protease inhibitors), respectively, may affect the metabolism of NPY in serum; 2) to assign kinetic values to the proteases involved in the cleavage process toward NPY; and 3) to characterize new NPY-truncated forms and to check for their possible binding capacities on NPY receptors.     

Biodegradation of all stereoisomers of the EDTA substitute iminodisuccinate by Agrobacterium tumefaciens BY6 requires an epimerase and a stereoselective C-N lyase

Biodegradation tests according to Organization for Economic Cooperation and Development standard 301F (manometric respirometry test) with technical iminodisuccinate (IDS) revealed ready biodegradability for all stereoisomers of IDS. The IDS-degrading strain Agrobacterium tumefaciens BY6 was isolated from activated sludge. The strain was able to grow on each IDS isomer as well as on Fe(2+)-, Mg(2+)-, and Ca(2+)-IDS complexes as the sole carbon, nitrogen, and energy source. In contrast, biodegradation of and growth on Mn(2+)-IDS were rather scant and very slow on Cu(2+)-IDS. Growth and turnover experiments with A. tumefaciens BY6 indicated that the isomer R,S-IDS is the preferred substrate. The IDS-degrading enzyme system isolated from this organism consists of an IDS-epimerase and a C-N lyase. The C-N lyase is stereospecific for the cleavage of R,S-IDS, generating d-aspartic acid and fumaric acid. The decisive enzyme for S,S-IDS and R,R-IDS degradation is the epimerase. It transforms S,S-IDS and R,R-IDS into R,S-IDS. Both enzymes do not require any cofactors. The two enzymes were purified and characterized, and the N-termini were sequenced. The purified lyase and also the epimerase catalyzed the transformation of alkaline earth metal-IDS complexes, while heavy metal-IDS complexes were transformed rather slowly or not at all. The observed mechanism for the complete mineralization of all IDS isomers involving an epimerase offers an interesting possibility of funneling all stereoisomers into a catabolic pathway initiated by a stereoselective lyase.     

Chemische Zusammensetzung und Oberflächenstruktur der epikutikularen Wachse bei verschiedenen Organen von Jojoba

Zusammenfassend soll festgehalten werden: Die Oberflächen von Blatt und Samenschale sind bei Jojoba mit vergleichbaren Wachsmengen überzogen. Die unterschiedlichen morphologischen Wachsstrukturen dieser Organe sind primär eine Funktion der chemischen Natur, der Zusammensetzung und der Verteilungsmuster ihrer epikutikularen Wachse. So sind die kristallinen Wachsstrukturen der Blätter mit ihren Wachsplättchen bedingt durch die Dominanz sehr langkettiger und gesättigter aliphatischer Verbindungen, vor allem den hohen Anteilen an freien Fettsäuren, Alkoholen und Wachsestern. Die flüssige Konsistenz der Wachsschicht bei Jojoba-Samenschalen ist vor allem begründet im Vorliegen von hohen Anteilen an ungesättigten Verbindungen bei Wachsestern, freien Fettsäuren und Sterinen, wie auch im Vorkommen von verzweigten Alkanen. Außerdem besitzen die meisten Substanzen der Samenwachse eine kürzere Kettenlänge als die der Blattwachse sowie abgeflachte Verteilungsmuster. Diese chemischen Daten verursachen eine Schmelzpunktdepression bei diesem Wachsgemisch mit der Folge, daß Jojoba-Samenschalen bei Zimmertemperatur mit einer flüssigen Wachsschicht überzogen sind. Die Ausführungen haben auch gezeigt, daß die verschiedenen Jojoba-Organe eine charakteristische und spezifische Zusammensetzung ihrer epikutikularen Wachse aufweisen und daher auch organspezifische Oberflächen-Feinstrukturen besitzen. Dies sind Befunde, die auch bei allen anderen untersuchten Pflanzen beobachtet werden konnten.