Tropinone is an alkamine intermediate at the branch point of biosynthetic pathways leading to various tropane alkaloids. Two stereospecifically distinct NADPH-dependent oxidoreductases, TR-I and TR-11, which, respectively, reduce tropinone to 3a-hydroxytropane (tropine) and 3j@-hydroxytropane (4-tropine), were detected mainly in the root of tropane alkaloid-producing plants but not in nonproducing cultured root. Both reductases were purified to near homogeneity from cultured root of Hyoscyamus niger and characterized. The TR-1 reaction was reversible, whereas the TR-11 reaction was essentially irreversible, reduction of the ketone being highly favored over oxidation of the alcohol 4-tropine. Marked differences were found between the two reductases in their affinities for tropinone substrate and in the effects of amino acid modification reagents. Some differences in substrate specificity were apparent. For example, N-propyl-4-piperidone was reduced by but not by TR-1. Conversely, 3-quinuclidinone and 8-thiabicyclo[3,2, 1]octane-3-one were accepted as substrates by TR-I but hardly at all by TR-11. Both enzymes were shown to be class B oxidoreductases, which transfer the pro-S hydrogen of NAD(P)H to their substrates. Possible roles of these tropinone reductases in alkaloid biosynthesis are discussed.Tropinone is an alkamine intermediate at the branch point of biosynthetic pathways leading to various tropane alkaloids mainly found in Solanaceae (Fig. 1). The 3-keto group of tropinone can be reduced to hydroxyls having two different configurations: tropine has a hydroxy group on the opposite side of the tropane nitrogen atom (a-configuration), whereas 0-tropine has the group on the same side (fl-configuration). (TR-II) was later found in cultured roots of Hyoscyamus niger (5). Subsequent studies by us (23) and others (4) indicated that the two stereospecifically distinct reductase activities can coexist in a given alkaloid-producing species. How the metabolic fates of tropinone are controlled by these reductases is an interesting question, particularly in view of our observation that the {-tropine-forming activity seemed to be predominant in cultured H. niger roots that accumulated mostly tropine-derived alkaloids (5). Here we report distribution of the two reductases in various plant species, their organspecific expression, and detailed biochemical characterization of the enzymes highly purified from cultured roots of H. niger. MATERIALS AND METHODS Plant MaterialsCultured roots of Hyoscyamus niger L. have been maintained in our laboratory since 1984 as described elsewhere (13). Several plant species (Physochlaina orientalis M.B. Don, Atropa acuminata Royle ex Lindl, Physalis alkekengi L., and Calystegia sepium R. Br) were transformed with Agrobacterium rhizogenes (strain 15834) by Dr. P. Bachmann at our laboratory. A hairy root clone (D15/5) of Datura stramonium was obtained from Dr. R. Robins, Institute of Food Research, Norwich, UK. These roots were cultured in auxin-free B5 medium (7) supplemented with ...

No abstract

No abstract

beta-(Isoxazolin-5-on-2-yl)alanine (BIA), a biosynthetic precursor of the neurotoxic amino acid beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), was confirmed to be derived from O-acetyl-L-serine (OAS) and isoxazolin-5-one by cysteine synthase in higher plants. Some properties of this enzyme in the biosynthesis of BIA are described.