5-Tetrahydroprotoberberine-oxidase

Based on knowledge of a biosynthetic pathway one can select certain steps which could be of interest for bioconversion of (a) readily available precursor(s). This could, for example, be stereospecific reactions, like the reduction of quinidinone in quinine or quinidine and the epoxidation of atropine to scopolamine. For the bioconversion one can consider using plant cells [e.g., the production of L-dopa from tyrosine by immobilized cells of Mucuna pruriens (10)] or isolated enzymes from the plant itself. An interesting example of the latter is the (5)-tetrahydroprotoberberine oxidase (STOX) enzyme, which oxidizes (5)-reticuline but not its stereoisomer (11). This feature can be used in the production of (i )-reticuline from a racemic mixture (see below). Immobilized strictosidine synthase has been successfully used to couple secologanin and tryptamine. The gene for this enzyme has been isolated from Rauvolfia (6) and cloned in Escherichia coli, in which it is expressed, resulting in the biosynthesis of active enzyme (7). The cultured bacteria produced 20 times more enzyme... 

An enzyme, (5)-tetrahydroprotoberberine oxidase, from suspension-cultured cells of Berberis wilsoniae catalyzes the removal of four hydrogen atoms from a number of tetrahy-droprotoberberine alkaloids in the presence of oxygen (Amann et al., 1988). 

Amann, M, N. Nagakura, and M. H. Zenk, Purification and properties of (5)-tetrahydroprotoberberine oxidase from suspension-cultured cells of Berberis wilsoniae, Eur. J. Biochem., 175, 17-25 (1988). 

Amann M, Nagakura N, Zenk MH. Purification and properties 52. of (S)-tetrahydroprotoberberine oxidase from suspension cultures... 

Gesell, A., et al.. Heterologous expression of two FAD -dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells. Planta, 2011. 233(6) p. 1185-97. 

The first reaction in the direction of the protoberberines is the formation of the berberine bridge (Fig. 2), catalyzed by the berberine bridge enzyme (abbreviated BBE), which specifically only accepts (S)-reticuline and not the (R)-enantiomer as substrate (ref. 23). As a next step a methylation at C-9 follows to form (S)-tetrahydrocolumbamine catalyzed by a methyltransferase with a much higher substrate specificity than the methyltransferases described before (ref. 24). In the genus Berberis (S)-tetrahydrocolumbamine is then oxidized by an enzyme called (S)-tetrahydroprotoberberine oxidase (abbreviated STOX) (ref. 25) and finally the methylene dioxy bridge is formed by the so called berberine synthase (ref. 26). 

M. Amann, N. Nagakura and M.H. Zenk, (S)-Tetrahydroprotoberberine oxidase, the final enzyme in protoberberine biosynthesis. Tetrahedron Lett. 25 (1984), 953-954. 

Fig. 4 Pathways of benzylisoquinoline biosynthesis. A selection of biosynthetic enzymes is notified with their localization in the cytosol (yellow), the ER membrane (red) or in the lumen of cytosolic vesicles (blue). Informations are taken mainly from Facchini and St-Pierre (2005), Bock et al. (2002), own experiments and other references cited in the text. BBE, berberine bridge enzyme CFS, cheilanthifoline synthase CNMT, coclaurine N-methyltransferase COR, codeinone reductase DBOX, dihydrobenzophen-anthridine oxidase MSH, N-methylstylopine 14-hydroxylase NCS, norcoclaurine synthase NMCH, N-methylcoclaurme 3 -hydroxylase 4 OMT, 3 -hydroxy-N-methylcoclaurine 4 -0-methyltransferase 60MT, norcoclaurine 6-O-methyltransferase 70MT, reticuline 7-O-methyltransferase P6H, protopine 6-hydroxylase SAT, salutaridinol-7-O-acetyltransferase SOR, salutaridineiNADPH 7-oxidoreductase STS, stylopine synthase SAS, salutaridine synthase TNMT, tetrahydroprotoberberine cis-N-methyltrans-ferase TYDC, tyrosine decarboxylase CAS, canadine sjmthase SOMT, scoulerine 9-O-methyltransferase STOX, (S)-tetrahydroprotoberberine oxidase...

This enzyme [EC 1.5.3.9], also called reticuline oxidase and tetrahydroprotoberberine synthase, catalyzes the re-... 

Berberine chloride shows anthelmintic activity and is able to eliminate Syphacia obvelata from mice. A series of synthetic 8-substituted tetrahydroprotoberberines has demonstrated both antifungal and antiarrhythmicactivity. Berberine sulfate and tetrahydropalmatine inhibit the respiratory chain by interfering with the action of NADH oxidase." ... 

Reticuline oxidase BBE berberine bridge enzyme berberine-bridge-forming enzyme tetrahydroprotoberberine synthase oxidoreductases Isoquinoline alkaloid biosynthesis (m00950)... 

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