Biosynthetic enzymes alkaloids

Facchini, P. J. and St.-Pierre, B. 2005. Synthesis and trafficking of alkaloid biosynthetic enzymes. Current Opinion in Plant Biology, 8(6) 657-666. 

The translocation of pathway intermediates is also suggested by differential localization of TLA biosynthetic enzymes. In situ hybridization and immunolocalization studies have shown that CYP72A1, TDC, and STR are localized to the epidermis of immature leaves, stems, and flower buds.52,147 In contrast, D4H and DAT are associated with laticifers and idioblasts of shoot organs. Laticifers and idioblasts are distributed throughout the mesophyll in C. roseus leaves, and are often several cell layers away from the epidermis. Vindoline biosynthesis involves at least two distinct cell types and requires the intercellular translocation of a pathway intermediate (Fig.7.9B). The differential localization of the early and late steps of vindoline biosynthesis might partially explain why this alkaloid is not produced in dedifferentiated C. roseus cell cultures. 

In summary, the pumiliotoxin-A class alkaloids are unique to certain genera from four different families of amphibians. Pumiliotoxins and allo-pumiliotoxins often occur together. Homopumiliotoxins occur much less often. The unique and complex structures and the phylogenetic distribution of pumiliotoxin-A class alkaloids argue for the independent evolutionary development of a complex suite of biosynthetic enzymes. The branched-chain structures of the pumiliotoxin-A class also suggest the incorporation of isoprene units, unlike the preponderance of bicyclic den-drobatid alkaloids, which appear to be derived from straight-chain precursors. 

Overproduction of berberine in C. japonica cell suspension cultures was achieved by selection of a high-producing cell line (34) with reported productivity of berberine reaching 7 g/L (35). This overproduction is one of the first demonstrations of production of a benzylisoquinoline alkaloid in cell culture at levels necessary for economic production. This cell line has facilitated greatly the identification of the biosynthetic enzymes. 

However, biosynthetic enzymes, which catalyze these reactions, have not been purified. Therefore, further studies are required to elucidate the effect of light on the formation and accumulation of the alkaloids. The contents of protoemetine, which have been regarded as the biosynthetic precursor of cephaeline and emetine, were extremely low irrespective of the different culture conditions. 

Facchini PJ, St-Pierre B. Synthesis and trafficking of alkaloid biosynthetic enzymes. 

Scheme 5. Biosynthesis of pyrrolizidine alkaloids. A molecular clone has been isolated for only one biosynthetic enzyme. Abbreviation HSS, homospermidine synthase.

Many alkaloid biosynthetic enzymes have been localized to subcellular compartments other than the cytosol 260). Enzyme compartmentalization sequesters toxic alkaloids and pathway intermediates away from sensitive areas of the cell. The subcellular trafficking of biosynthetic intermediates might also create an important level of metabolic regulation. Understanding the subcellular compartmentalization of alkaloid pathways will show whether enzyme characteristics observed in vitro represent bona fide regulatory mechanisms in vivo. 

Figure 2.8 Production of alkaloids by enzymatic conversion of amino acids in plant cell, yeast, and E. coli by transfer of plant biosynthetic enzyme genes into these microorganisms, from [63]. MAO overexpressed mono-amine oxidase from E. coli, NCS norcoculaurine synthase, MTs specific O- and N-methyltransferases, BBE berberine bridge-forming enzyme, CYP80G2 diphenyl ring bridging enzyme.

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. 

Additional studies are needed for the improvement of tropane alkaloids production in a scale useful for commercialization. However, the overexpression of several key biosynthetic enzymes is a promising strategy for the improvement of secondary metabohtes production. 

Genomic and transcriptomic technologies have been used to rapidly identify biosynthetic steps. There are currently over 40,000 expressed enzyme tags (ESTs) generated fi om alkaloid-producing plants that have been used to isolate genes involved in the alkaloid pathway [7]. Some alkaloid biosynthetic steps occur as spontaneous chemical reactions without the use of enzymes, for example, conversion of the intermediate neopine into codeinone in the morphine biosynthetic pathway. Also, some enzymes may catalyze two or more separate reactions in the pathway, for example, hyoscyamine 6-hydroxylase, which carries out two consecutive steps in the scopolamine biosynthetic pathway. Alkaloid biosynthesis also involves compartmentalization. Tissue-specific localization studies have shown that sequential biosynthetic enzymes can occur in distinct cell types [8, 9]. During the biosynthesis of the indole alkaloids vinblastine and vincristine in Catharanthus roseus, different enzymatic steps are carried out in different cellular compartments (Fig. 8.5) [10]. Various steps in the pathway are carried out in different types of cell. This requires the intercellular transport of metabolic intermediates. Similarly, scopolamine biosynthesis also involves two different cell types. 

Table 8.2 Alkaloid biosynthetic enzymes for which the corresponding genes have been cloned (Adopted from Ref. [3]) ...

---