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In vitro studies with a microsomal system obtained from etiolated sorghum seedlings have indicated a biosynthetic pathway for cyanogenic glucosides involving amino acids, N-hydroxyamino acids, aldoximes, nitriles and cyanohydrins. NADPH is an essential cofactor. Simultaneous measurements of tyrosine metabolism and oxygen consumption show that three molecules of oxygen are consumed for each molecule of p-hydroxymandelonitrile produced. This indicates the operation of three monooxygenases in the pathway and implies the involvement of one hitherto undetected intermediate in the pathway. The nature of this intermediate is unknown. Gel filtration and sucrose gradient centrifugation of the microsomal system resulted in a more than tenfold increase in specific activity. Attempts to further purify the system did not produce preparations of higher specific activity because of a simultaneous partial loss of essential components as demonstrated by reconstitution experiments.

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Pure bromoperoxidase from the marine alga, Penicillus capitatus, converts alpha-amino acids and peptides to the corresponding decarboxylated nitriles and aldehydes in the presence of hydrogen peroxide and bromide ion. Thus, both valine and valylvaline are converted to isobutyronitrile and isobutyraldehyde while alanine is converted to acetonitrile and acetaldehyde. The reaction is nonstereospecific and can be catalyzed by bromoperoxidases obtained from different sources. Bromoperoxidase catalyzes the conversion of methoxytyrosine to p-methoxyphenylacetonitrile. This reaction is consistent with the involvement of bromoperoxidase in the formation of aeroplysinin-1, a brominated aromatic nitrile antibiotic produced by a marine sponge.

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The last step in the biosynthesis of cyanogenic glucosides, the glucosylation of the cyanohydrin intermediate, has been investigated in detail using Triglochin maritima seedlings. The glucosyltransferase activity is not associated with membranes and appears to be a "soluble" enzyme. The cyanohydrin intermediate, which is formed by hydroxylation of 4-hydroxyphenylacetonitrile by a membrane-bound enzyme, is free to equilibrate in the presence of the glucosyltransferase and UDPG, because it can be trapped very efficiently. This indicates that this intermediate is not channeled (unlike some of the other intermediates), although it is probably the most labile of all of them. The glucosyltransferase of T. maritima responsible for the glucosylation of the cyanohydrin was separated from another glucosyltransferase, which used 4-hydroxybenzylalcohol as a substrate, and purified over 200-fold. It catalyzed the glucose transfer from UDPG to only 4-hydroxymandelonitrile and 3,4-dihydroxymandelonitrile, giving rise to the respective cyanogenic glucosides. Although the activities with these two substrates behaved differently in certain respects (e.g., extent of inactivation during purification and difference in activation by higher salt concentrations), most of the data acquired favor the view that only one enzyme in T. maritima is responsible for the glucosylation of both substrates.

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The in vitro biosynthesis of the cyanogenic glucoside taxiphyllin has recently been demonstrated in Triglochin maritima (Hösel, W., and Nahrstedt, A. (1980) Arch. Biochem. Biophys. 203, 753-757). We have now studied in more detail the multistep conversion of tyrosine into p-hydroxymandelonitrile, the immediate precursor of taxiphyllin, catalyzed by microsomes isolated from dark-grown seedlings. The biosynthetic pathway involves N-hydroxytyrosine, p-hydroxyphenylacetaldoxime, and p-hydroxyphenylacetonitrile. In marked contrast to an analogous pathway in Sorghum bicolor, p-hydroxyphenylacetonitrile is the best substrate for cyanide production (Vmax = 224 nmol/h/g, fresh wt) and the physiological substrate tyrosine is the poorest (Vmax = 18.8 nmol/h/g, fresh wt). The substrates exhibit alkaline pH optima between 7.5 and 9, and all except tyrosine show pronounced substrate inhibition. We have found that p-hydroxyphenylacetonitrile generated in situ from tyrosine is free to equilibrate by diffusion with exogenous material. On the other hand, neither N-hydroxytyrosine nor p-hydroxyphenylacetaldoxime will readily exchange with exogenous intermediates. We consider both N-hydroxytyrosine and p-hydroxyphenylacetaldoxime to be channeled in T. maritima, whereas in S. bicolor N-hydroxytyrosine and p-hydroxyphenylacetonitrile are channeled and the aldoxime is freely exchangeable.

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The biosynthetic pathway for the cyanogenic glucoside, dhurrin, involves the following intermediates: L-tyrosine, N-hydroxytyrosine, p-hydroxyphenylacetaldoxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile. N-Hydroxytyrosine and p-hydroxy-phenylacetonitrile produced from L-tyrosine by microsomes from seedlings of Sorghum bicolor are utilized more effectively as substrates than exogenously added N-hydroxytyrosine and p-hydroxyphenylacetonitrile. The minimum values for the channeling ratios are 25 for N-hydroxytyrosine and 115 for p-hydroxyphenylacetonitrile. On the other hand, p-hydroxyphenylacetaldoxime produced internally exchanges readily with exogenously added p-hydroxyphenylacetaldoxime. These results indicate that the biosynthetic pathway is catalyzed by two mutienzyme complexes or by two multifunctional proteins and explain why the rate of the overall sequential reaction starting from L-tyrosine is greater than the rates of reaction initiated later in the sequence with the known intermediates N-hydroxytyrosine and p-hydroxyphenylacetonitrile. Attempts to cross-link chemically the last enzyme in the pathway, a soluble UDP-glucose glucosyl-transferase, to the microsomal system were unsuccesful.

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A32390A is an isonitrile-containing derivative of diacyl D-mannitol. The compound is produced in fermentation as the major component of a metabolic complex known as A32390. A32390A inhibits dopamine-beta-hydroxylase reduces heart and adrenal norepinephrine levels, lowers blood pressure in hypertensive rats, and possesses antibiotic activity vs. Gram-positive bacteria and fungi, including Candida albicans. A32390 is produced in submerged culture by a mold, a species of Pyrenochaeta, NRRL-5786. Glucose and sucrose are among the best carbon sources for the biosynthesis of A32390. Mannitol, although a substituent of the A32390A molecule, supports little or no biosynthesis of the compound when employed as the major carbon source for the fermentation. The addition of crotonic acid derivatives. ethanol, or L-histidine to the fermentation medium enhances the level of A32390 produced.

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Dhurrin, I ((S)-p-hydroxymandelonitrile-beta-D-glucopyranoside), and taxiphyllin, II (the (R) epimer), occur in the genera Sorghum and Taxus, respectively. Both derive biosynthetically from L-tyrosine via the hydroxylation of p-hydroxyphenylacetonitrile, III. (3R)- and (3S)-L-[3-3H1]tyrosine, prepared by enzymic hydroxylation of the corresponding phenylalanines, were fed separately to shoots from sorghum seedlings (Sorghum bicolor (Linn) Moench) and cuttings from Japanese Yew (Taxus cuspidata Sieb. and Zucc.) and the appropriate cyanogenic glycoside was isolated (I or II). The fraction of the 3H conserved in I and II was calculated from both parallel feeding and 3H:14C double labeling experiments. The results for II were the reverse of I. Both hydroxylations of III, which give rise to the enantiomeric products (S)- and (R)-p-hydroxymandelonitrile, occurred with retention of configuration. The retention mode is characteristic of hydroxylations at aliphatic carbons catalyzed by mixed function oxidases.

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