Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

Dopamine produces vasolidation in specific regions and layers of the rabbit gastrointestinal tract

The effect of intravenous dopamine infusion (25 and 60 μg per kg and min consecutively) on blood flow distribution in the splanchnic region of anesthetized rabbits was studied applying the microsphere technique. During infusion of the low dose, blood flow increased most markedly in the stomach, less in the pancreas, jejunum and descending colon, and decreased in the spleen. In the stomach the increase was confined to the mucosa-submucosa. Raising the dose of dopamine resulted in a slight fall of arterial blood pressure, a further increase in blood flow through the mucosa-submucosa of the gastric fundus (+493 % as against control), but not through the other tissues studied. In another series, blood flow through the left gastric artery was measured with an electromagnetic flowmeter. The infusion of dopamine produced a dose-dependent increase in regional blood flow, which was inhibited by the dopamine antagonist bulbocapnine. Furthermore, the control blood flow was transiently decreased, and resistance to flow was increased by bulbocapnine. The results indicate that the dopamine-induced vasodilation in the gastrointestinal tract of the rabbit is largely restricted to the gastric circulation and suggest that specific receptors mediating this vasodilation are located in the mucosa-submucosa. It is hypothesized that endogenous dopamine functions as a vasodilatory tissue hormone in the gastric mucosa of the rabbit.     

Types of sesquiterpene-coumarin ethers from Achillea ochroleuca and Artemisia tripartita

In addition to known derivatives, four new sesquiterpene-coumarin ethers were isolated from the roots of Achillea ochroleuca and Artemisia tripartita and identified by ¹H and ¹³C NMR spectroscopy, including lanthanide induced shifts. The new compounds are isofraxidin derived ethers which differ from the previously described derivatives by ring cleavage and methyl migration within the terpenoid unit. The chemosystematic importance of sesquiterpene-coumarin ether accumulation within the two genera is briefly discussed.     

Biosynthesis of cyanogenic glucosides: in vitro analysis of the glucosylation step

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.     

Allosteric activation of the hydrolysis of specific substrates by chymotrypsin.

A variety of azobenzene compounds having bis-quaternary nitrogens have been shown to accelerate the hydrolysis by chymotrypsin of certain specific substrates by an allosteric mechanism. One of the most potent, 2,2'-bis[alpha-(benzyldimethylammonium)methyl]azobenzene dibromide (2,2'-QBzl) accelerated the hydrolysis of glutaryl-L-phenylalanine p-nitroanilide 40-fold at saturating concentration. Acceleration was by increasing kcat without altering Km. The hydrolysis of acetyl-L-tyrosine p-nitroanilide and acetyl-L-tyrosine anilide was also accelerated by Q-Bzl (25-fold and 1.8-fold respectively) while the hydrolysis of hemoglobin, azocoll and a number of esters was not affected. The inactivation of chymotrypsin by diphenylcarbamyl chloride and diphenylcarbamyl fluoride was accelerated by 2,2'-Q-Bzl. Reac;ivation in the presence of NH2OH was also accelerated, but in the absence of added nucleophile (i.e. of NH20H) no increase in rate was detectable. An allosteric effector was covalently attached to chymotrypsinogen A by reaction with 2,2'-bis[alpha-(o-bromomethylbenzyldimethylammonium)methyl]azobenezene dibromide. The product, when converted to active enzyme, was about 4 times more active than chymotrypsin as a result of an increase in kcat of hydrolysis; Km was unaffected. The mechanism of the allosteric acceleration process is not known but, because for all of the substrates affected acylation of the enzyme is rate-limitimg, it is tentatively suggested that the effectors facilitate proton transfer to the leaving group by an inductive effect on the 'charge relay system'. Spectral studies indicate that the allosteric site is a portion of the enzyme with a polarity near that of water, possibly on the outside surface of the enzyme molecule.