Functional Analysis of Norcoclaurine Synthase in Coptis japonica

Abstract

(S)-Norcoclaurine is the entry compound in benzylisoquinoline alkaloid biosynthesis and is produced by the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS) (EC 4.2.1.78). Although cDNA of the pathogenesis-related (PR) 10 family, the translation product of which catalyzes NCS reaction, has been isolated from Thalictrum flavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isolated from Coptis japonica (CjNCS1) also catalyzed NCS reaction as well as a PR10 homologue of C. japonica (CjPR10A). Both recombinant proteins stereo-specifically produced (S)-norcoclaurine by the condensation of dopamine and 4-HPAA. Because a CjNCS1 cDNA that encoded 352 amino acids showed sequence similarity to 2-oxoglutarate-dependent dioxygenases of plant origin, we characterized the properties of the native enzyme. Sequence analysis indicated that CjNCS1 only contained a Fe 2؉ -binding site and lacked the 2-oxoglutarate-binding domain. In fact, NCS reaction of native NCS isolated from cultured C. japonica cells did not depend on 2-oxoglutarate or oxygen, but did require ferrous ion. On the other hand, CjPR10A showed no specific motif. The addition of o-phenanthroline inhibited NCS reaction of both native NCS and recombinant CjNCS1, but not that of CjPR10A. In addition, native NCS and recombinant CjNCS1 accepted phenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, as well as 4-HPAA, for condensation with dopamine, whereas recombinant CjPR10A could use 4-hydroxyphenylpyruvate and pyruvate in addition to the above aldehydes. These results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C. japonica cells was more active and formed a larger complex compared with recombinant CjNCS1.Higher plants produce divergent chemicals such as alkaloids, terpenoids, and phenolic compounds in secondary metabolism. Among these chemicals, alkaloids are very important in medicine because of their high biological activities. Alkaloids are low molecular weight, nitrogen-containing compounds that are found in ϳ20% of plant species. Most alkaloids are derived from amines produced by the decarboxylation of amino acids such as histidine, lysine, ornithine, tryptophan, and tyrosine. Although the coupling of amines to other products is the first important step in producing diverse alkaloids, this entry reaction has been poorly characterized. (S)-Strictosidine is a central intermediate for indole alkaloids. Strictosidine synthase, which catalyzes the formation of (S)-strictosidine from tryptamine and secologanin, is a rare exception in that its cDNA has been cloned from Catharanthus roseus and Rauvolfia serpentine (1, 2).Benzylisoquinoline alkaloids are a large and diverse group of pharmaceutical alkaloids with ϳ2,500 defined structures. Norcoclaurine produced from tyrosine is a key entry compound from which various benzylisoquinoline alkaloids such as analgesic morphine, colchicines, antibacterial berberine...

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…The stereoselective Pictet-Spengler condensation of (S)-norcoclaurine from 4HPAA and dopamine is catalyzed by norcoclaurine synthase (NCS), which is a member of the pathogenesis-related (PR)10/Bet v 1 protein family (Liscombe et al 2005;Samanani et al 2004;Lee and Facchini 2010). In addition to a PR10-type NCS, a second enzyme (CjNCS1) sharing similarity with ODDs was reported to catalyze the formation of (S)-norcoclaurine in Coptis japonica (Minami et al 2007). However, the actual physiological role of this enzyme is not known.…”

Strictosidine Synthase: Mechanism of a Pictet−Spengler Catalyzing Enzyme

“…Enzymes that catalyze this reaction have been isolated from several plant alkaloid biosynthetic pathways. [1][2][3] Strictosidine synthase, 4,5 one "Pictet-Spenglerase," is the central enzyme in the biosynthesis of the monoterpene indole alkaloids. These alkaloids are a large and structurally diverse family of natural products that include vinblastine, yohimbine, ajmaline, camptothecin, and strychnine and are found in a wide variety of plant species.…”

Characterization of Three O-Methyltransferases Involved in Noscapine Biosynthesis in Opium Poppy

“…Among these, opium poppy (Papaver somniferum) remains a valuable source for several pharmaceuticals, including the narcotic analgesics morphine and codeine, the muscle relaxant papaverine, and the cough suppressant and potential anticancer drug noscapine. BIA biosynthesis begins with the formation of (S)-norcoclaurine via the condensation of the Tyr derivatives dopamine and 4-hydroxyphenylacetaldehyde (Samanani and Facchini, 2002;Minami et al, 2007;Lee and Facchini, 2010). (S)-Norcoclaurine is subsequently converted via several steps to the key branch point intermediate (S)-reticuline ( Fig.…”