Tropinone reductase

Tropinone reductase-I Hyoscyamius niger Datura stramonium... 

Tropinone reductase-II Hyoscyamius niger Datura stramonium Solanum tuberosum... 

Richter, U., Rothe, G Fabian, A. K., Rahfeld, B. and Drager, B. 2005. Overexpression of tropinone reductases alters alkaloid composition in Atropa belladonna rootcultures. Journal of Experimental Botany, 56(412) 645-652. 

Keiner, R., Kaiser, H., Nakajima, K., Hashimoto, T., Drager, B. (2002). Molecular cloning, expression and characterization of tropinone reductase II, an enzyme of the SDR family in Solanum tuberosum (L.). Plant Mol Biol, 48,299-308. 

NAKAJIMA, K., HASHIMOTO, T., Two tropinone reductases, that catalyze opposite stereospecific reductions in tropane alkaloid biosynthesis, are localized in plant root with different cell-specific patterns. Plant Cel Physiol., 1999,40, 1099-1107. 

Figure 2.3 Biosynthesis of the tropane alkaloids. PMT, putrescine N-methyltransferase DAO, diamine oxidase MPO, N-methylputrescine oxidase TR I and II, tropinone reductase H6H, hyoascyamine 6-hydroxylase.

Tropinone reductases, which catalyse the stereospecific reduction of the keto group of tropinone to 3a- and 3 3-hydroxy groups (Drager, 2005), were analysed in detail by dissection of the peptides and construction of chimeric enzymes. The opposite stereospecificity of the two reductases was ascribed to the carboxy-half of the proteins, to which the substrate tropinone is assumed to bind with the reverse orientation in the two enz)mries (Nakajima et al, 1993, 1994 Hashimoto and Yamada, 1994). Only tropinone with the 3a-hydroxy group is used to produce hyoscyamine (Leete, 1990). Tropinone with the 3 3-hydroxy group forms esters with other acids, but these occur only as minor alkaloids. When tril was overexpressed in A. belladonna root cultures, a substantial amount of pseudotropine and related alkaloids was observed (Richter et al, 2005). 

Drager, B. (2005) Tropinone reductases, enzymes at the branch points of tropane alkaloid metabolism. Phytochemistry, 67, 327-37. 

Drager, B., Portsteffen, A., Schaal, A., McCabe, P.H., Peerless, A.C.J. and Robins, R.J. (1992) Levels of tropinone reductase activities influence the spectrum of tropane esters found in transformed root cultures of Datura stramonium. Planta, 188, 581-6. 

Kaiser, H., Richter, U., Keiner, R., Brabant, A., Hause, B. and Drager, B. (2006) Im-munolocalisation of two tropinone reductases in potato Solanum tuberosum L.) root, stolon, and tuber sprouts. Planta, 225, 127-37. 

Nakajima, K., Hashimoto, T. and Yamada, Y. (1993) cDNA encoding tropinone reductase-II from Hyoscyamus niger. Plant Physiol., 103,1465-6. 

Nakajima, K., Yamashita, A., Akama, H., Nakatsu, T., Kato, H., Hashimoto, T., Oda, J. and Yamada, Y. (1998) Crystal structures of two tropinone reductases different reaction stereospecificities in fhe same protein fold. Proc. Natl. Acad. Sci. USA, 95, 4876-81. 

Tropanone then is reduced via an NADPH-dependent reductase to tropine that has been cloned from Hyoscyamus niger (149, 150). All tropane-producing plants seem to contain two tropinone rednctases, which create a branch point in the pathway. Tropinone reductase I yields the tropane skeleton (Fig. 3b), whereas tropinone rednctase II yields the opposite stereocenter, pseudotropine (151). Tropane is converted to scopolamine or hyoscyamine, whereas the TRII product pseudotropine leads to calystegines (152). These two tropinone reductases have been crystalhzed, and site-directed mutagenesis studies indicate that the stereoselectivity of the enzymes can be switched (153, 154). 

Figure 3 (a) Representative tropane and nicotine alkaloids, (b) Tropane biosynthesis. ODC, ornithine decarboxylase PMT, putrescine N-methyltransferase MPO, diamine oxidase TRl, tropinone reductase 1 H6H, hyocyamine 6b-hydroxylase. 

Hashimoto T, Nakajima K, Ongena G, Yamada Y. Two tropinone reductases with distinct stereospecificities from cultured roots of Hyoscyamus niger. Plant Physiol. 1992 100 836-845. 

Rocha P, Stenzel O, Parr A, Walton NJ, Christou P, Drager B, Leech MJ. Functional expression of tropinone reductase I and hyoscyaine-6b-hydroxylase from Hyoscyamus niger in Nicotiana tabacum. Plant Sci. 2002 162 905-913. 

Nakajima K, Kato H, Oda J, Yamada Y, Hashimoto T. Site directed mutagenesis of putative substrate binding residues reveals a mechanism controlling different substrate specificities of two tropinone reductases. J. Biol. Chem. 1999 274 16563-16568. Yamashita A, Kato H, Wakatsuki S, Tomizaki T, Nakatsu T, Nakajima K, Hashimoto T, Yamada Y, Oda J. Structure of tropinone reductase-II with NADP + and pseudotropine at 1.9A resolution implication for stereospecific substrate binding and catalysis. Biochemistry 1999 38 7630-7637. 

F. (1). Tropane alkaloids biosynthetic pathway. The known enzymes are indicated. ODC (ornithine decarboxylase), ADC (arginine decarboxylase), PMT (putrescine methyl transferase), MPO (methyl putrescine oxidase), TRI, TRII (tropinone reductase I, II), H6H (hyoscyamine 6p hydroxylase). 

Tropinone is stereospecifically reduced to yield both tropine (3a-hydroxytropine), which led to the formation of tropane alkaloids, and pseudotropine (3p-hydroxytropine), the precursor of calystegines. These stereospecific reductions are catalyzed by two different tropinone reductases, tropinone reductase I and II (TRI and TRII). Both enzymes have been isolated from many Solanaceous species. Thus, TR I and TR II were isolated from D. innoxia roots and the crude extract favoured the production of pseudotropine over tropine [151]. Also, from transformed root cultures of D. stramonium two different tropinone reductases were obtained. In this species, TRI showed about 5-fold larger activity than TRII, and TRI displayed a pronounced pH-dependency, while TRII was more tolerant to different pH values [152]. Moreover, two tropinone reductases were also isolated from H. niger root cultures. TRI-reduction was reversible, whereas TRII-reduction was essentially irreversible [153]. In subsequent studies it was found that the accumulation of both TRs was the highest in the lateral roots of H. niger throughout development, with different cell-specific patterns [154]. 

Tropinone is reduced stereospecifically to either tropine or pseudotropine (Fig. 4). This reduction is brought about by two independent tropinone reductases, often referred to as TR-I and TR-II [30], TR-I catalyzes the NADPH-dependent formation of tropine, whereas TR-II reduces tropinone to pseudotropine. The TR-I reaction is reversible but the TR-II reaction is essentially irreversible, the reduction of the ketone being highly favoured over the oxidation of the alcohol pseudotropine [31]. Results of feeding indicate that 3a-tropine and 3(3-tropine do not isomerize and that only the former is incorporated into hyoscyamine [32]. 

Tropinone reductase-l Atropa belladonna Hyoscyamius niger... 

Nakajima K, Hashimoto T, Yamada N. Two tropinone reductases with different stereospecificities are short-chain dehydrogenases evolved from a common ancestOT. Proc Natl Acad Sci USA 1993 90 9591-5. 

A key branch point in tropane alkaloid biosynthesis is the conversion of tropinone into either tropine or pseudotropine (or i/ -tropine), which possess opposite stereochemistry at the 3-hydroxyl position. Two different NADPH-dependent enzymes catalyze the stereospecilic reduction of tropinone the 3-carbonyl of tropinone is reduced to the 3a-hydroxy group of tropine by tropinone reductase I (TR-I) and to the 3jS-hydroxy group of pseudotropine by tropinone reductase II (TR-II). Genes encoding both TR-I and TR-II have been identified in the tropinone... 

Scheme 3. Biosynthesis of the tropane alkaloids hyoscyamine and scopolamine, the ca-lystegines, and nicotine. Molecular clones have been isolated for the enzymes shown. Abbreviations CYP82E4, nicotine A-demethylase H6H, hyoscyamine 6jS-hydroxylase ODC, ornithine decarboxylase PMT, putrescine A-methyltransferase TR-I, tropinone reductase-I TR-II, tropinone reductase-II.

The use of undifferentiated cultures proved to be unsuccessful for tropane alkaloids production. These alkaloids are produced in normal and transformed roots [42, 51]. Several lines of evidence suggest that the differentiation of the tissue is necessary for the synthesis of these metabolites [42, 51]. Different studies suggest that this is probably related to the localization of key biosynthetic enzymes [6]. Among them, Suzuki et al. [52, 53] demonstrated that the h6h and pmt genes were expressed specifically in root pericycle of Atropa belladonna plants. In addition, Nakajima et al. [54, 55] pointed out that Tropinone reductase enzymes were accumulated in lateral roots of Hyoscyamus niger. 

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