Norcoclaurine 6-0-methyltransferase

Fig. 10.8 Selected cDNAs isolated in recent years that encode enzymes involved in the biosynthesis of various classes of isoquinoline alkaloids. 6-OMT, norcoclaurine 6-0-methyltransferase 23 CYP80A1, berbamunine synthase 19 CYP80B1, (S)-A-methylcoclaurine 3 -hydroxylase 20 CPR, cytochrome P-450 reductase 29 4 -OMT, (5)-3 -hydroxy-A-methylcoclaurine 4 -0-methyltransferase 30 BBE, berberine bridge enzyme 12 SalAT, salutaridinol 7-O-acetyltransferase 28 COR, codeinone reductase.25...

Scheme 1. Biosynthesis of the benzylisoquinoline alkaloids berberine, morphine, and sang-uinarine. Enzymes for which corresponding molecular clones have been isolated are shown in bold. Abbreviations 4 OMT, 3 -hydroxy-A-methylcoclaurine 4 -0-methyltransferase 60MT, norcoclaurine 6-0-methyltransferase 70MT, reticuline 7-0-methyltransferase BBE, berberine bridge enzyme CFS, cheilanthifoline synthase CNMT, coclaurine A-methyltransf-erase COR, codeinone reductase CYP719A1, canadine synthase CYP80A1, berbamunine synthase CYP80B1, A-methylcoclaurine 3 -hydroxylase DBOX, dihydrobenzophenanthri-dine oxidase DRR, 1,2-dehydroreticuline reductase DRS, 1,2-dehydro reticuline synthase MSH, A-methylstylopine 14-hydroxylase NCS, norcoclaurine synthase P6H,...

Oimaroon, A., et al, (R,S)-Reticuline 7-0-methyltransferase and (R,S)-norcoclaurine 6-0-methyltransferase of Papaver somniferum - cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. Plant J, 1003. 36(6) p. 808-19. 

BIA biosynthesis starts with the two tyrosine condensation product 4-hydroxyphenyl acetaldehyde (4-HPAA) and dopamine catalyzed by norco-claurine synthase (NCS) (Figure 6.19) [93, 94]. The so-formed (5)-norcoclaurine represents the first pathway intermediate with a benzylisoquinoline skeleton from which the BIA classes are derived. Methylation of the latter by norcoclaurine 6-0-methyltransferase (6-OMT) leads to (5)-coclaurine, which is methylated by coclaurine A-methyltransferase (CNMT) to yield A-methylcoclaurine. Hydrox-ylation of the 3 position, catalyzed by (5)-A-methylcoclaurine 3 -hydroxylase,... 

The conversion of (5)-norcoclaurine to (i )-reticuline, the immediate precursor to oxidative cyclization, clearly requires three methylations ( ), an oxidation, and an isomerization (the latter effected by an oxidation and then a reduction). Thus, as shown in Scheme 13.41, it has been found that (i 5)-norcoclaurine 6-0-methyltransferase (EC 2.1.1.128) effects the methylation of (S)-norcoclaurine through transfer of a methyl group from 5-adenosylmethionine to phenolic hydroxyl at C-6 of (5)-norcoclaurine generating (5)-coclaurine and 5-adenosylhomocysteine. Then, a second methylation, with the same methylation source but now under the influence of the enzyme (5)-coclaurine-A -methyltransferase (EC 2.1.1.140) produces (5)-A -methylcoclaurine. 

Fig. 8.12 Reconstruction of benzylisoquinoline alkaloid (BIA) pathway in microorganisms. MAO bacterial monoamine oxidase from Micrococcus luteus, NCS norcoclaurine synthase from Coptis japonica, 6-OMT norcoclaurine 6-0-methyltransferase from Coptis japonica, CNMT, coclaurine-]V-melhyltransferase from Coptis japonica, 4-OMT 3 -hydroxy-iV-methylcoclaurine-4 -0-methyltransferase from Coptis Japonica, CYP80G2 plant cytochrome P450 enzymes from Coptis japonica, BBE berbeiine bridge enzyme from Coptis japonica (Adopted from Ref. [101])...

Fig. 18 Reconstructed isoquinoline alkaloid biosynthetic pathway in microbes (E. coli and S. cerevisiae) [120]. Accession numbers of the genes can be taken from Gene Bank. C3,4-DHPA-A 3,4-dihydroxyphenylacetaldehyde NCS norcoclaurine synthase 60MT 6-0-methyltransferase CNMT coclaurine-/V-mcthyltransferase 4 OMT 4 -0-methyltransferase BBE berberine bridge enzyme NMT /V-methyltransferase)...

The three cDNAs of norcoclaurine-6-O-methyltransferase, coclaurine-AA methyltransferase and 3 -hydroxy-AAmethylcoclaurine 4,-<9-methyltransferase converting (R, S)-norlaudanosoline into (R, Sj-reticuline with a 5-10% yield... 

Ounaroon, A., G. Decker, J. Schmidt, F. Lottspeich and T.M. Kutchan, (R,S)-Reticuline 7-O-methyltransferase and (R,S)-Norcoclaurine 6-O-Methyltransferase of Papaver... 

With the assumption that reticulines are also precursors in mammalian synthesis of morphine, it was challenging to investigate whether they could be produced by enzymatic reactions similar to those utilized in benzylisoquinoline-producing plants (274). This plan focused attention on reactions controlled by the enzyme catechol 0-methyltransferase (COMT), using 5-adenosyl-L-methionine (SAM) for the methylation reaction. Mammalian COMT is present in mammalian tissues, particularly the liver, and an enzyme preparation from rat liver was used for the experiments. It was found that (S)-norcoclaurine, which is the first isoquinoline produced in benzylisoquinoline-producing plants, was similarly O-methylated in vitro by SAM in the presence of COMT, and a reverse proportion of methylated products was obtained with the (/ )-enantiomer (277). Similar 0-methylation of (5)-4 -demethylreticuline (3 -hydroxy-N-methylcoclaurine), prepared by total synthesis (162), however, afforded almost exclusively (5)-orientaline, with a methoxy group at C-3 and not at C-4 as in (5)-reticuline (Fig. 37) (762). 

Figure 2.5 Formation of (5)-reticuline. NCS, norcoclaurine synthase 60MT, 6-hydroxy-O-methyltransferase CNMT, coclaurine N-methyltransferase 40MT, 4 hydroxy-0-methyltransferase.

Sato F, Tsujita T, Katagiri Y, Yoshida S, Yamada Y. Purification and characterization of S-adenosyl-L-methionine norcoclaurine 6-O-methyltransferase from cultured Coptis japonica cells. Eur. J. Biochem. 1994 225 125-131. 

The three steps of methylation of norlaudanosoline (iii, iv, v) are catalyzed by three kinds of methyltransferases (60MT, CNMT, and 4 OMT). These enzymes need 5-adenosyl-L-methionine (SAM) as the methyl donor. The 60MT and CNMT from C. japonica have broad substrate selectivity. Therefore, 60MT can catalyze the 6-(9-methlyation of not only norcoclaurine (VI) but also norlaudanosoline (iii). Similarly, CNMT can catalyze the N-methylation of not only coclaurine (VII) but also 6-(9-methylnorlaudanosoline (iv). 

Fig. 8.6 Biosynthetic pathway for benzylisoquinoline alkaloid (BIA) biosynthesis in plants. TYDC tyrosine/dopa decarhoxylase, 60MT norcoclaurine b-O-methyltransfoase, 40OMT 30-hydroxy-Af-methylcoclaurine 40-O-methyltransferase, OMT ITl O-methyltransferase II-l, CYP80A1 berbamunine synthase, CYP80B1 (Si-N-methylcoclaurine 30-hydroxylase, btaber-ine bridge enzyme, SOMT scoulerine iV-methyltransferase, COR codeinone rednctase (Adopted from Ref. [3])...

The isoquinoline pathway s enzyme orthologs in other plants showed that genes of methyltransferase family such as (R,S)-reticuline 7-0-methyltransferase, coclaurine 4 -0-methyltransferase, (S)-norcoclaurine 6-0-methyltrasferase, columbamine 0-methyltransferase, coclaurine A-methyltransferase, putrescine A-methyltransferase responsible for the production of reticuline, coclaurine, norcoclaurine, columbaine have been reported from Papaver somniferum, Coptis japonica, Thalictrum flavum, Thalictrum tuberosum, Coffea liberica, Cojfea arabica, Cojfea canephora, Nicotiana tabacum. Solarium tuberosum. Datura stramonium, Hyoscyamus niger, and Atropa belladonna. Pathway contains information of about four alkaloids such as dopamine, colchicine, ephedrine, and methamphetamine and identified two missing links. 

Figure 4.2 Biosynthesis of benzylisoquinoline alkaloids. Enzyme abbreviations TYDC, tyrosine/dopa decarboxylase NCS, norcoclaurine synth lse 60MT, norcoclaurine 6-O-methyltransferase 4 OMT, 3 -hydroxy-/V-methylcoclaurine 4 -0-methyltransferase CYP80A, berbamunine synth lse CYP80B, N-methylcoclaurine 3 -hydroxylase BBE, berberine bridge enzyme SOMT, scoulerine 9-0-methyltransferase CYP719A, (S)-canadine synthase SAT, salutaridinol 7-O-acetyltransferase COR, codeinone reductase.

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