Volume 42, Issue 4

Stereochemical studies on tyrosine phenol-lyase from Escherichia intermedia have shown that the α,β-elimination reactions of ʟ-serine and ᴅ- and ʟ-tyrosine proceed with retention of config­uration at C-β. Stereospecifically (β-tritiated ʟ-serine is slowly racemized at C-β Deuterium from the α-position of ʟ-tyrosine is partially transferred to C-4 of the phenol formed when the α,β- elimination reaction is carried out in H 2 O, although no transfer of α- 1 H in 2 H 2 O was seen. The result favors tautomerization of the p-hydroxyphenyl to a cyclohexadienonyl moiety prior to carbon-carbon bond cleavage. In the conversion of ʟ- to ᴅ-alanine catalyzed by tyrosine phenol- lyase, some a-hydrogen recycling is observed, pointing to a single-base racemization mechanism. Attempts to demonstrate cofactor motion during racemization by NaBH 4 reduction of [ 3 H]PLP- enzyme: ᴅ- and ʟ-alanine complexes failed, but showed that, as in other PLP enzymes, the holoenzyme is reduced preferentially from the Re face with respect to C-4′ of PLP and enzyme- substrate complexes preferentially from the Si face.

The incorporation rates of labelled tyrosine, DOPA. tyramine. and dopamine have been inves­tigated during the in vivo formation of the protoberberine alkaloid, jatrorrhizine, in callus cul­tures of Berberis canadensis. While tyrosine was equally well incorporated into both the iso­quinoline (54%) and benzyl (46%) portions of the alkaloid, DOPA was almost exclusively (91%) transformed into the isoquinoline moiety. However, tyramine (25%) and to a lesser extent, dopamine (15%) were incorporated into the aldehyde-derived, benzylic half of the isoquinoline molecule as well. In order to investigate further the precursory roles of these compounds, select enzymes involved in tyrosine metabolism in alkaloid-producing cell cultures have been studied. The occurrence of tyrosine decarboxylase, phenolase, transaminase, p-hydroxyphenylpyruvate decarboxylase, amineoxidase and methionine adenosyl transferase was demonstrated in suspen­sion cells of Berberis. These enzymes were partially purified and a preliminary characterization was performed. In the light of these and previous data, the differential metabolism of tyrosine and DOPA in the early steps of isoquinoline alkaloid biosynthesis is discussed. Conclusive evidence as to the biosynthetic origin of the phenylacetaldehydes which furnish the benzylic moiety of the alkaloids is precluded by the presence of both amineoxidase and phenylpyruvate decarboxylase activities in these cultures.

A novel enzyme was isolated, partially purified (217-fold) and characterized from cell suspen­sion cultures of Rauwolfia serpentina Benth. The enzyme catalyzes one of the late biochemical reactions in the biosynthesis of ajmaline by hydrolysis of 17-O-acetylated alkaloids of the ajmalan group forming the appropriate deacetylated compounds. This esterase exhibits an unusually high substrate selectivity and exclusively accepts acetylated ajmaline derivatives with the naturally occurring 2β(R)-configuration. The properties of the enzyme were determined showing an optimum pH at 7.5, an isoelectric point of pH 4.9 and a relative molecular weight of 33 ± 2 kDa. Inhibition studies of enzyme activity point to the necessity of SH-groups. The esterase seems not to be inhibited by ajmaline. the end product of the pathway. The highest enzyme activities were observed in leaves and cell suspension tissues of the tribe Rauwolfieae which are known to synthe­size ajmaline and its congeners. The specific function of the esterase in the biosynthesis of the later alkaloids was established.

Microsomal preparations from osmotically stressed soybean cells catalyze the conversion of (2S)-naringenin to apigenin in presence of NADPH. In contrast, such preparations from normal soybean cells or from elicitor-challenged cells catalyze the conversion of (2S)-naringenin to genistein (isoflavone synthase). It is concluded that osmotic stress of the cells causes a switch from isoflavone to flavone synthesis. The flavone synthase from osmotically stressed cells corresponds in its properties to the microsomal flavone synthase found in several flowers (G. Stotz and G. Forkmann, Z. Naturforsch. 36c, 737-741 (1981)) and differs from the flavone synthase I from parsley cell cultures which is a soluble Fe 2+ and 2-oxoglutarate dependent dioxygenase. Flavone synthase II from soybean has an absolute requirement for NADPH and oxygen. It is inhibited by carbon monoxide in presence of oxygen and this inhibition is reversed by light. It is also inhibited by cytochrome c and by a number of cytochrome P-450 inhibitors. This and other properties show that flavone synthase II is a cytochrome P-450 dependent monooxygenase.

Incubation of urocanase with 2-methylurocanate leads, after an initial normal reaction, to a time dependent inactivation of the enzyme. It is suggested that a tautomeric form (1) of the product, 2-methyl-imidazolone propionate, is the actual inhibitor. On the basis of these and of published experimental data a novel mechanism is proposed for the urocanase reaction. The crucial and initial step is the electrophilic addition of enzyme-bound NAD to the 2-position of the imidazole nucleus of urocanate.

We have purified diol dehydrase, an adenosylcobalamin-dependent enzyme, from Klebsiella pneumoniae by two different procedures to re-investigate its protein structure; one including its extraction with detergent from the membrane fraction, and the other consisting of only chromato­graphic separations of the soluble fraction. The enzyme preparations obtained by these two methods were different in the subunit structure, but both are identical in molecular weight, and in enzymological and immunochemical properties. In addition, the enzyme preparation obtained from the membrane fraction dissociated reversibly into two dissimilar protein components (F and S) in the absence of substrate, as did the preparation from the soluble fraction. Although the subunit multiplicity of component S might be partly due to proteolytic cleavage during the enzyme purification as revealed by limited digestion with trypsin, component F is not a product of proteolytic cleavage of component S, but a primordial and essential constituent of the enzyme.

Cell suspensions of Methanosarcina barkeri grown on acetate catalyze the formation of methane and CO 2 from acetate as well as an isotopic exchange between the carboxyl group of acetate and CO 2 . Here we report that these cells also mediate the synthesis of acetate from methyl iodide, CO 2 , and reducing equivalents (H 2 or CO), the methyl group of acetate being derived from methyl iodide and the carboxyl group from CO 2 . Methyl chloride and methyltosylate but not methanol can substitute for methyl iodide in this reaction. Acetate formation from methyl iodide, CO 2 , and reducing equivalents is coupled with the phosphorylation of ADP. Evidence is pres­ented that methyl iodide is incorporated into the methyl group of acetate via a methyl corrinoid intermediate (deduced from inhibition experiments with propyl iodide) and that CO 2 is assimi­lated into the carboxyl group via a C 1 intermediate which does not exchange with free formate or free CO. The effects of protonophores, of the proton-translocating ATPase inhibitor N.N′-di- cyclohexylcarbodiimide, and of arsenate on acetate formation are interpreted to indicate that the reduction of CO 2 to the oxidation level of the carboxyl group of acetate requires the presence of an electrochemical proton potential and that acetyl-CoA or acetyl-phosphate rather than free acetate is the immediate product of the condensation reaction. These results are discussed with respect to the mechanism of methanogenesis from acetate.

The organization of genes for 5S rRNA in the methanogenic archaebacterium Methanococcus (M .) voltae and their nucleotide sequences have been determined. M. voltae possesses three 5S rRNA genes, one of them is organized in an rRNA transcriptional unit coding for 16S-23S-5S rRNA. The other two are associated with seven tRNA genes in a putative transcriptional unit composed of 5′-tRNA Thr -tRNA Pro -tRNA Tyr -tRNA Lys - 5S rRNA-tRNA Asp -tRNA Lys - 5S rRNA-tRNA Asp -3′. Coding regions plus spacers of the tRNA Lys -5S rRNA-tRNA Asp block of this gene cluster occur twice with identical sequence. The 5S rRNA from this cluster displays considerable sequence divergence to the rRNA operon-linked 5S rRNA gene. Comparison of the M. voltae 5S rRNA sequences with those from M. vannielii revealed that the operon-linked genes on one hand and the tRNA-linked 5S genes on the other share a greater sequence homology than the two types of genes within each of the two organisms. This indicates an independent evolution of the two sets of 5S rRNA genes without selective pressure from other ribosomal components or, alternatively, lateral gene transfer.

Three ergoline alkaloids producing Claviceps strains easily formed protoplasts when treated with snail digestive extract ex Helix pomatia. Colonies obtained after regeneration of protoplasts were monitored for alkaloid production. Among the regenerated progeny strains could be isolated which differ considerably in the quantity of ergolines and the composition of the alkaloid mixture compared with parental strains. Protoplast-derived strains were stable in morphology and alkaloid production for more than four years if the propagation was performed with hyphal fragments. Activities of some alkaloid-specific enzymes e.g. oxygenases and ergotamine syntheta­se were studied in various related strains.

The enzymes of the terminal steps of phenylalanine and tyrosine biosynthesis, chorismate mutase, prephenate dehydratase, arogenate dehydratase, prephenate dehydrogenase and aroge­ nate dehydrogenase were studied in 13 sporeforming members of the order Actinomycetales. In these organisms tyrosine is synthesized exclusively via arogenate, phenylalanine, however, via phenylpyruvate. The regulation pattern of the corresponding enzymes was determined: No feed­ back inhibition of arogenate dehydrogenase by L-phenylalanine and ʟ-tyrosine was observed. Chorismate mutase was found to be inhibited in all organisms by ʟ-tyrosine and in most organisms by ʟ-tryptophan. ʟ-Phenylalanine was shown to inhibit prephenate dehydratase in the majority of bacteria tested and ʟ-tyrosine activated this enzyme in most cases. The elution profiles for the phenylalanine and tyrosine biosynthetic enzymes were studied in three members of the order Actinomycetales by anion exchange chromatography on DEAE-cellulose.

The complete 796 residue amino acid sequence of maltodextrin phosphorylase was deduced from the E. coli malP nucleotide sequence. The calculated molecular weight of 90 500, including pyridoxal phosphate, is significantly larger than experimentally determined values. Enzymatically active and inactive mutants following deletion or exchange of up to 8 codons (7 amino acids) at the 3′ end (C-terminus) confirm the size of the mature native enzyme and disclose the essential functional or structural role of the highly conserved C-terminal region of phosphorylases.

In the absence of oxygen Bacillus macerans is able to ferment xylose. The principal products are ethanol, formate,CO 2 , acetone and H 2 . The yield of ethanol was 61% based on the theoretical value of 0.51 g per g xylose consumed. The cells could grow in the presence of up to 4% (v/v) of added ethanol but the growth rate was already reduced to about 50% by 1% (v/v) of alcohol. Glucose and the pentoses arabinose and xylose were sequentially utilized when initially present as a mixed substrate. Enzymatic studies indicate that xylose was metabolized via the pentosephos-phate and Embden-Meyerhof pathways. At an oxygen concentration of 1-2% air saturation 42 g/l xylose were completely fermented within 160 h and 10 g/l ethanol were produced.

The citric acid excretion of Ca-alginate-immobilized cells of Aspergillus niger in batch culture decreased with a half-time of approximately 19 days. Reactivation of the biocatalysts by regeneration in growth medium was possible, but it was followed by a submerged sporulation of the fungus, and medium was highly contaminated with free cells. Citric acid production could better be prolonged by semicontinuous cultivation with medium exchange every 7 or 14 days, respectively. After 32 days the remaining activity in semicontinuous culture was 1.4-fold higher than in comparable batch experiments. Similar improvements were obtained with a continuous process at a dilution rate of 0.125 v/v · d, whereby medium efflux completely free of detaching mycelia.

Biotransformation of cedrol with 5 different strains afforded 8 previously undescribed hydroxy-cedrols. The attack came from a hemisphere centered at C-12 approximately. At two positions epimeric pairs were formed, which could be avoided by using different strains. Contrary to previous studies of other groups we found the 2-and 12-hydroxylations as main reactions. Cedrene needed prolonged fermentation and gave lower yields than cedrol. Apart from allylic hydroxylations the oxidation pattern by Corynespora cassiicola DSM 62474 of cedrene and cedrol was the same. The structures of the metabolites were elucidated by 2D NMR techniques.

6-Methylpurine (1), 6-methyl-9-β-ᴅ -ribofuranosylpurine (2) and 6-hydroxymethyl-9-β-ᴅ -ribo- furanosylpurine (3) were isolated from mycelial cultures of Collybia maculata and their antifungal, cytotoxic and antiviral activities investigated. This is the first report on the natural occurrence of these compounds.

The condensation of 3-hydroximino-2-butanone (1) with 5-amino-6-ribitylamino-2,4(1H,3H)- pyrimidinedione (2) yields 6,7-dimethyl-8-ribityllumazine (3). At slightly alkaline pH, the car­bonyl group of 1 reacts preferentially with the 5-amino group of 2 (regioselectivity, 4:1). Under acidic conditions, the reaction occurs with higher yield and marginal regioselectivity of opposite direction (1:1.4). Appropriately 13 C-labeled samples of 1 afford 3 labeled at C-6α, C-6, C-7 or C-7α. [6α,7α- 13 C 2 ]-3 was prepared by condensation of 2 with [1,4- 13 C 2 ]diacetyl. The lumazines 3 were converted to riboflavin by the enzyme, riboflavin synthase, with almost quantitative yield. By this procedure, any C-atom of the carbocyclic moiety of riboflavin can be selectively labeled with 13 C at high abundance. Phosphorylation yields the respectively 13 C-labeled FMN samples.

The grass Trisetum flavescens (golden oat grass, Goldhafer) causes soft tissue calcification in cattle and in sheep. The calcinogenic principle of the plant is the active vitamin D steroid hormone 1,25-Dihydroxyvitamin D 3 , the major physiological regulator of calcium homeostasis in higher animals. From comparison with synthetic vitamin D metabolites in different bioassays, it is concluded that T. flavescens contains the 25-glucoside of 1,25(OH) 2 D 3 . This compound, or rather the 1,25(OH) 2 D 3 liberated by ruminal fluid, is the calcinogenic factor of the grass.

The pheromone components of A. polyphemus, (6 E, 11 Z)-6 ,11-hexadecadienyl acetate and (6 E, 11 Z)-6 ,11-hexadecadienal, both effective also in A. pernyi, were synthetically varied in their chemical structures and these compounds electrophysiologically tested in single cell recordings. It appeared that for the interaction between the signal molecule and the receptor region at the dendritic membrane of the receptor cell the electronic character of the functional end group (acetate or aldehyde) of the stimulus molecule is important. The excitation of the sensory cell also depends on the chain length.

5′-Dimethoxytrityl-3′-phosphite amides of deoxynucleosides are synthesized. Phosphite/phosphate is protected by the 2,2,2-trichloro-1,1-dimethyl-ethyl (TCB) group, heterocyclic bases by the 2,2,2-trichloro-2,2-dimethyl-ethoxycarbonyl (TCBOC) group. Deoxyguanosine is also block­ ed by 6-O-trichloroethyl thus avoiding the difficulties observed with monoprotected guanine residues.

A series of 1-amino-2-cyclohexene-3-ols is subjected to an enzymatic transesterification by the system lipase on silica/tributyrin (Klibanov-method). Thereby, an optical resolution of 1r-benzoyl-amido-5t,6c-dimethoxy-1-cyclohexene-4c-ol is achieved. Advantages and disadvantages of this method are discussed.

(R)-and S)-4-methyl-[1- 3 H,2- 2 H 1 ]pentanal were prepared from l -and D-leucine via leucic acid and (S)-and (R)-4-methyl-[2- 2 H 1 ]pentanoic acid. Decarbonylation of these samples with tris-(triphenylphosphine)rhodium chloride followed by Kuhn-Roth oxidation of the resulting 2-methylbutane gave chiral acetic acid of 35% e.e. S and 31% e.e. R configuration, respectively. The decarbonylation reaction thus proceeds with net retention of configuration, possibly accom­panied by some racemization.

Polystyrene-polyoxyethylene craft copolymers have been used for step-wise peptide synthesis. After completion of synthesis the protecting groups are cleaved under acidic conditions, where the polymer-peptide bond is stable. These gels in comparison to polystyrene peptide gels, show better properties for applications in affinity chromatography as well as synthesis on solid supports, because the advantageous properties of polystyrene beads are combined with the excellent spacer behavior of polyoxyethylene chains (mobility, solvation by water and organic solvents). Peptide gels with polylysine sequences have been synthesized as highly selective stationary phases for the separation of the homologous oligo desoxyribonucleotides (dT) n with n = 1-3. The principal possibilities of these gels for affinity chromatography is demonstrated.

Normal engraftment of bone marrow transplants depends on histocompatibility between donor and recipient. In this case reconstitution of hemopoiesis and lymphopoiesis in the mouse spleen is correlated with transient biopterin synthesis in these cells. Allogeneic bone marrow progeny cells undergo donor cell proliferation in the spleen prior to the incidence of graft-versus-host reaction. These cells are not committed to biopterin synthesis. Thy-1 monoclonal antibody fully repairs engraftment of allogeneic transplants together with biopterin synthesis.

Glutathione (GSH) adducts and consecutive degradation products thereof are indications of reactive intermediates during drug metabolism. As demonstrated with the analgesic SX-PP 16 (4-amino-3,5-dibromacetanilide), however, interactions of a drug with GSH can be detected by labelling the GSH-stores with labelled cysteine, and consecutive administration of the unlabelled drug even at therapeutic doses. The GSH-adducts are sensitively and specifically traced by HPLC, applying column-switching and a combination of diode array-and radioactivity detection. This approach seems to be much more sensitive than a classical GSH-depletion study. The structure of the main metabolite of SX-PP 16 (46% of urinary excretion) was elucidated as 3-bromo-4-amino-5-mercapturyl-acetanilid.

The synthesis of a ditopic linear receptor 3 consisting of an azacrown ether moiety for binding prim, ammonium functions and a macrotricyclic tetrahedral moiety for binding hydrophobic guest species is described. The covalent interconnection of these subunits was supposed to constitute a receptor with enhanced specificity towards dopamine-type biogenic amines. Host-guest complexa­tion was studied in a competition method with potassium ion using ion selective electrodes. The binding features of 3 with a series of biogenic amines reveal that neither the absolute binding strength nor the selectivity is enhanced in relation to the monotopic receptor 4. It is concluded that 3 does not simultaneously utilize both receptor subunits in binding the biogenic amine substrates.

The metabolism of the herbicide [ 14 C]2-(2,4-dichlorophenoxy)-propionic acid (dichlorprop) was studied in excised seedlings of barley (Hordeum vulgare). It was rapidly taken up and metabolized by the plants yielding 5 metabolites. The metabolites, isolated by extraction with aqueous acetone, separated and purified by TLC, were identified by enzymatic, chemical, and spectrometric methods. Hydroxylation takes place at the 4-position of the aromatic ring, involving the NIH-shift of the chlorine to the 5-position and, to a minor extent, to the 3-position. The parent compound and also the 4-hydroxy derivatives undergo conjugation with mono- and diglucoses at the carboxyl and at the hydroxyl group, respectively. The time course of metabolism during 24, 48, and 72 h is presented, indicating interconversion reactions.

Three synthetic oligopeptides were used for preparation of antibodies against the D-2 polypeptide of thylakoid membranes. Their sequence was chosen from a model of the folding of the amino acid sequence of the D-2 polypeptide subunit through the membrane that predicted these sequences to be exposed at the membrane surface. For the Merrifield solid-phase method on a fully automated synthesizer the N a -amino group was protected by a fluorenyl-9-methylcarbonyl group. The oligopeptides were coupled to serum albumin by EDAC for immunizations in rabbits. Antisera with high titer were obtained for the two oligopeptides that contained the amino acid sequence of the D-2 protein from amino acid 230 to 235 and from 235 to 241. The antisera reacted with the D-2 polypeptide, separated on SDS gel and agglutinated the thylakoid membrane. It is known that certain photosystem II functions are impaired by short time trypsin treatment of the membrane. The antisera were used to show that under these conditions the D-2 polypeptide in the membrane is very sensitive. The trypsination yielded two cleavage products detected by the two antisera, a 20 kDa fragment blotted by antiserum against amino acids 230 to 235 and a 10 kDa fragment blotted by the antiserum against amino acids 235 to 241. As the polypeptide cleavage occurs between the two epitopes, the trypsin cut is therefore at arginine 234. This supports the prediction that the sequence containing this arginine is the most exposed part of the D-2 polypeptide on the membrane (matrix) surface. It is proposed that the high sensitivity of the D-2 polypeptide accounts for the known effect of membrane trypsination on Q A accessibility in photosystem II.