225 The expanding universe of alkaloid biosynthesis Vincenzo De Luca*† and Pierre Laflamme* Characterization of many of the major gene families responsible developments in our understanding of selected alkaloid- for the generation of central intermediates and for their biosynthesis pathways and provides a basic framework for decoration, together with the development of large genomics rapid progress in the study of the biological roles played by and proteomics databases, has revolutionized our capability to these small molecules in plants. More generally, the stage identify exotic and interesting natural-product pathways. Over is clearly set for the rapid characterization of whole biosyn- the next few years, these tools will facilitate dramatic advances thetic pathways for the diversity of plant-derived small in our knowledge of the biosynthesis of alkaloids, which will far molecules that give specific plants their characteristic surpass that which we have learned in the past 50 years. aroma, flavor, toxicity and pharmacological properties. These tools will also be exploited for the rapid characterization This rapid expansion of knowledge should then lead to of regulatory genes, which control the development of unlimited biotechnological applications for the production specialized cell factories for alkaloid biosynthesis. of improved crops that have enhanced nutritional and/or health-promoting properties. Addresses *Institut de Recherche en Biologie Végétale, Département de Biosynthesis of central intermediates Sciences Biologiques, Université de Montréal, 4101 rue Sherbrooke est, Most alkaloids are derived through the decarboxylation of Montréal, Québec, H1X 2B2, Canada amino-acid precursors (i.e. ornithine, lysine, tyrosine, tryp- †Present address: Syngenta Agribusiness Biotechnology Research Inc., 3054 Cornwallis Road, Research Triangle Park, North Carolina tophan, and histidine) to yield their respective amines, or 27709-2257, USA; e-mail: [email protected] from anthranilic acid or nicotinic acid. The ability of plants to couple amines to different chemical partners produces a Correspondence: Vincenzo De Luca restricted number of versatile chemical backbones (i.e. Current Opinion in Plant Biology 2001, 4:225–233 central intermediates) from which the diversity of alkaloids 1369-5266/01/$ — see front matter is produced. For example, molecules such as strictosi- © 2001 Elsevier Science Ltd. All rights reserved. dine, norcoclaurine, 1,3-dihydroxy-N-methylacridone and homospermidine are central intermediates in the synthesis Abbreviations of monoterpenoid indole (Figure 1a), isoquinoline ACS acridone synthase BPF-1 Box P Binding Factor-1 (Figure 1b), acridine (Figure 1c) and pyrrolizidine CHS chalcone synthase (Figure 1d) alkaloids, respectively. In fact, strictosidine and DAT deacetylvindoline-4-O-acetyltransferase norcoclaurine appear to be particularly versatile sources of D4H deacetylvindoline-4-hydroxylase diversity: over 50% of the more than 12,000 characterized DHS deoxyhypusine synthase HSS homospermidine synthase alkaloids are derived from these molecules. JA jasmonic acid MAT minovincinine-19-O-acetyltransferase The genes for some of these key reactions have been MIA monoterpenoid indole alkaloid cloned. Strictosidine synthase1 (Str1), which was cloned from OMT O-methyltransferase ORCA octadecanoid-derivative-responsive Catharanthus roseus R. serpentina and C. roseus, catalyses the formation of stric- APETALA2-domain tosidine from tryptamine and the monoterpenoid PAGE polyacrylamide gel electrophoresis secologanin. Strictosidine-synthase-like genes have PCR polymerase chain reaction recently been identified in animals (including humans), PNAE polyneuridine aldehyde esterase RG raucaffricine-O-β-glucosidase and there are at least three Str1-like genes in soybean and SGD strictosidine β-D-glucosidase 13 such genes in Arabidopsis thaliana (reviewed in [1]). In Str1 Strictosidine synthase1 Drosophila, the cell-surface protein hemomucin is com- T16H tabersonine 16-hydroxylase posed of a mucin-type repeat domain and an Str1-type TDC tryptophan decarboxylase domain. Hemomucin, which binds Helix pomatia lectin, may activate the immune response and may also be Introduction involved in haemolymph coagulation. The presence of The toxicity of plants, which contributes to their ability to Str1-like genes in A. thaliana and soybean, which are not protect themselves against predation, is partially related to known to produce indole alkaloids, suggests that Str1-like the diversity of small molecules that they synthesize. genes have other basic biological roles that precluded the Alkaloids, which display a large variety of pharmaceutical evolution of Str1 activity in indole-alkaloid biosynthesis. activities, compose one of the major classes of small mole- cules and accumulate in about 20% of all plant species. The key reaction in the biosynthesis of all acridone alka- Although the importance of alkaloids in medicine has been loids, which are restricted to some genera of the Rutaceae, highly publicized, the regulation of alkaloid biosynthesis, is catalyzed by acridone synthase (ACS). This enzyme, and the sites of alkaloid production and accumulation, is which converts N-methylanthraniloyl-CoA and 3 malonyl- only now being elucidated. This review highlights recent CoAs into 1,3-dihydroxy-N-methylacridone, has a function 226 Physiology and metabolism Figure 1 Examples of monoterpenoid alkaloids: (a) an N indole, (b) an isoquinoline, (c) an acridine and O (d) a pyrrolizidine. N + H N N O H3CO2C OCH H 3 H CO N OAc 3 H CO CH OCH H3C HO 2 3 3 (a) Anhydrovinblastine: (b) Berberine: fungitoxic, antineoplastic agent antibacterial O OCH3 HO O O O O N O CH3 H N (c) Rutacridone: fungitoxic, (d) Scenecionine: hepatotoxic, antibacterial insect pheromone precursor, antileukemic Current Opinion in Plant Biology that is similar to that of chalcone synthase (CHS; EC participates in the activation of the protein factor 5A, 2.3.1.74). Unlike CHS, however, ACS will not accept which is required for the activation of cell proliferation in 4-coumaroyl-CoA as a substrate. The initial isolation of an animal cells. Substrate specificity studies involving recom- ACSI clone (reviewed in [2•]), and the subsequent charac- binant tobacco DHS, S. vernalis DHS and S. vernalis HSS terization of the tandemly arranged and 94% homologous confirmed that DHS catalyses both the aminobutylation of ACSII gene [3], revealed how the ACS genes are evolu- putrescine and the activation of factor 5A, whereas HSS tionarily related to the CHS gene family and classified only uses putrescine as an aminobutyl acceptor [5]. The them as plant polyketide synthases. existence of DHS in S. vernalis [5] and in tobacco [6] sug- gests its involvement in processes similar to those found in Most of the 360 known pyrrolizidine alkaloids [4•] are animals, and that the HSS of alkaloid-producing species found within a scattered and restricted set of species within was most likely recruited from this primary biological the Asteraceae, Boraginaceae, Fabaceae and Orchidaceae. process. Selection pressure through herbivory may have Their scattered distribution within the angiosperms sug- been responsible for the evolution of an enzyme that does gests that the biosynthesis pathway of the pyrrolizidine not activate protein factor 5A but retains the ability to alkaloids may have arisen independently several times. transfer an aminobutyl moiety from spermidine to Pyrrolizidine alkaloids are strong feeding deterrents for putrescine. The presence of the DHS gene, and hence the most herbivores and are highly toxic to vertebrates causing availability of homospermidine precursors for pyrrolizidine damage to the liver. alkaloid synthesis, throughout the plant kingdom may explain why this pathway has successfully evolved several Pyrrolizidine alkaloids contain a characteristic necine base times [7•]. that is derived from putrescine and the aminobutyl moiety of spermidine, both of which are derived from arginine. Monoterpenoid indole alkaloid biosynthesis Homospermidine synthase (HSS; EC 2.5.1.44), which cat- Assembly of secologanin alyzes the formation of homospermidine from putrescine Secologanin provides the C9–C10 moiety in the biosyn- and spermidine, has recently been cloned from Scenecio thesis of monoterpenoid indole alkaloids (MIAs) in vernalis [5]. The remarkable amino-acid identity between C. roseus. In spite of extensive studies of the enzymes that HSS from S. vernalis and deoxyhypusine synthase (DHS; convert geraniol into secologanin in the C. roseus model EC 1.1.1.249) from S. vernalis (79%), tobacco (80%) and system, little is known about the genes involved. Recent humans (61%), strongly suggests that HSS evolved from 13C-glucose-feeding experiments with C. roseus cell cul- DHS. Both HSS and DHS transfer an aminobutyl moiety tures, showed that secologanin may be derived via the from spermidine to their individual substrate in a NAD+- triose phosphate/pyruvate pathway [8]. Secologanin is specific fashion. In contrast to HSS, however, DHS also formed via an initial rate-limiting hydroxylation of geraniol The expanding universe of alkaloid biosynthesis De Luca and Laflamme 227 by geraniol-10-hydroxylase, which leads to the formation Although we do not know how this reactive dialdehyde is of 10-hydroxygeraniol. Although geraniol-10 hydroxylase, channeled into each of the major alkaloid types, the timing a cytochrome P450 monooxygenase, was first purified to and site of SGD expression, together