CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin. JAUME BASTIDA (87), Departament de Productes Naturals, Facultat de Farma` cia, Universitat de Barcelona, 08028 Barcelona, Spain YEUN-MUN CHOO (181), Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia PETER J. FACCHINI (1), Department of Biological Sciences, University of Calgary, Calgary, AB, Canada TOH-SEOK KAM (181), Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia RODOLFO LAVILLA (87), Parc Cientı´fic de Barcelona, Universitat de Barcelona, 08028 Barcelona, Spain DANIEL G. PANACCIONE (45), Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6108, USA CHRISTOPHER L. SCHARDL (45), Department of Plant Pathology, University of Kentucky, Lexington, KY 40546-0312, USA PAUL TUDZYNSKI (45), Institut fu¨r Botanik, Westfa¨lische Wilhelms Universita¨tMu¨nster, Mu¨nster D-48149, Germany FRANCESC VILADOMAT (87), Departament de Productes Naturals, Facultat de Farma` cia, Universitat de Barcelona, 08028 Barcelona, Spain vii PREFACE This volume of The Alkaloids: Chemistry and Biology is comprised of four very different chapters; a reflection of the diverse facets that comprise the study of alkaloids today. As awareness of the global need for natural products which can be made available as drugs on a sustainable basis increases, so it has become increas- ingly important that there is a full understanding of how key metabolic pathways can be optimized. At the same time, it remains important to find new biologically active alkaloids and to elucidate the mechanisms of action of those that do show potentially useful or novel biological effects. Facchini, in Chapter 1, reviews the significant studies that have been conducted with respect to how the formation of alkaloids in their various diverse sources are regulated at the molecular level. The history of the ergot alkaloids and their biological effects is a very rich and fascinating one. In Chapter 2, advances in the biosynthetic pathway of these alkaloids are discussed, the genes involved are reviewed, and their biological and clinical significance are presented by Schardl, Panaccione, and Tudzynski. In Chapter 3, Bastida, Lavilla, and Viladomat provide a classical review of the isolation, structure elucidation, biosynthesis, synthesis, and biology of the Narcissus alkaloids. This group of alkaloids is receiving a great deal of attention at the present time because of the significant biological activities observed for some of these metabolites. It has been over 25 years since the broad area of the bisindole alkaloids was reviewed in this series. In the concluding chapter, Kam and Choo have taken on this formidable task and provide a very comprehensive summation of tremendous advances in the structural, synthetic, and biological aspects of more than 30 basic alkaloid types represented. Geoffrey A. Cordell University of Illinois at Chicago ix —CHAPTER 1— REGULATION OF ALKALOID BIOSYNTHESIS IN PLANTS PETER J. FACCHINI Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada I. Introduction II. Alkaloid Biosynthetic Pathways III. Regulation of Alkaloid Biosynthesis IV. Applications of Genomics to the Study of Alkaloid Biosynthesis V. Metabolic Engineering of Alkaloid Pathways VI. Future Prospects Acknowledgments References I. Introduction Several key technical breakthroughs over the last half-century have contrib- uted to an impressive advancement in our understanding of alkaloid biosynthesis in plants. The use of radiolabeled precursors in the 1950s allowed the elucidation of several biogenic pathways. The widespread application of plant cell cultures during the 1970s provided a rich source of biosynthetic enzymes and encouraged work on the elucidation of signal transduction mechanisms that activate alkaloid pathways. The introduction of molecular techniques in the 1990s prompted the isolation of numerous molecular clones involved in alkaloid biosynthesis (1), which have been used to de- termine the tissue-specific localization of alkaloid biosynthetic enzymes and gene transcripts, and functionally analyze the corresponding promoters. Recent applica- tions of genomics-based technologies, such as expressed sequence tag (EST) databas- es, DNA microarrays, and proteome analysis, have shown the potential to accelerate the discovery of new components and mechanisms involved in the assembly and function of plant alkaloids. Our emerging ability to investigate alkaloid metabolism from a combined biochemical, molecular, cellular, and physiological perspective has greatly improved our appreciation for the complex regulation of diverse biosynthetic pathways. Unlike other types of secondary metabolites, the different structural cat- egories of alkaloids have unique biosynthetic origins. This review will focus on recent advances in our understanding of the metabolic regulation involved in the biosyn- thesis of six groups of alkaloids – benzylisoquinoline, monoterpenoid indole, tropane, purine, pyrrolizidine, and quinolizidine alkaloids – for which biosynthetic and reg- ulatory genes have been reported and characterized. THE ALKALOIDS, Vol. 63 Copyright r 2006 Elsevier Inc. 1 ISSN: 1099-4831 All rights reserved DOI: 10.1016/S1099-4831(06)63001-0 2 FACCHINI II. Alkaloid Biosynthetic Pathways A. BENZYLISOQUINOLINE ALKALOIDS Many benzylisoquinoline alkaloids (BAs) are used as pharmaceuticals due to their potent pharmacological activity, which is often an indication of their biological function. For example, the effectiveness of morphine as an analgesic, colchicine as a microtubule disrupter, and (+)-tubocurarine as a neuromuscular blocker suggests that these alkaloids function as herbivore deterrents. The antimicrobial properties of sanguinarine and berberine suggest that they confer protection against pathogens (2). BAs occur mainly in basal angiosperms, including the members of Ran- unculaceae, Papaveraceae, Berberidaceae, Menispermaceae, and Magnoliaceae. BA biosynthesis begins with the conversion of tyrosine into both dopamine and 4–hydroxyphenylacetaldehyde by a lattice of decarboxylations, ortho-hydro- xylations, and deaminations (3). The aromatic amino acid decarboxylase (TYDC) that converts tyrosine and dopa into their corresponding amines has been purified, and several cDNAs have been cloned (Scheme 1) (4–6). A family of 15 genes, which can be divided into two subgroups based on sequence identity, encodes TYDC in opium poppy (Papaver somniferum) (5). The catalytic properties of the various protein isoforms are similar despite the differential developmental and inducible expression of the TYDC gene family (5,7–9). Dopa- mine and 4–hydroxyphenylacetaldehyde are condensed by norcoclaurine synthase (NCS) to yield (S)-norcoclaurine (Scheme 1), the central precursor to all BAs in plants (10,11). Owing to the inability of NCS to discriminate between 4–hydroxy- phenylacetaldehyde and 3,4–dihydroxyphenylacetaldehyde, and the nonspecificity of the subsequent methyltransferase reactions, it was originally thought that the tetrahydroxyBA (S)-norlaudanosoline was the precursor (12). However, only nor- coclaurine has been found to occur in plants. NCS has been purified (13,14) and corresponding molecular clones have been isolated from Thalictrum flavum (15) and P. somniferum (16). NCS is ancestrally related to the pathogenesis-related (PR) 10 and Bet v 1 protein families, which include the enzyme (HYP1) from St. John’s wort (Hypericum perforatum) responsible for the biosynthesis of the bioactive naphthodianthrone hypericin (17). The novel catalytic functions of NCS and HYP1 define a new class of plant secondary metabolic enzymes. (S)-Norcoclaurine is converted into (S)-reticuline by a 6–O-methyltransferase (6OMT), an N-methyltransferase (CNMT), a P450 hydroxylase (CYP80B1), and a 40-O-methyltransferase (40OMT) (Scheme 1) (18–21). 6OMT and 4’OMT were purified (18), and corresponding cDNAs isolated and characterized from Coptis japonica (22), T. flavum (23),andP. somniferum (24–26). Each enzyme exhibits unique substrate specificity and a different reaction mechanism despite extensive homology. A previ- ously reported family of catechol O-methyltransferases that accepts (S)-norcoclaurine as a substrate is probably not involved in alkaloid biosynthesis (27). CNMT has also been purified (28), and the corresponding cDNA isolated from C. japonica (29), T. flavum (23),andP. somniferum (24). The aromatic ring hydroxylation involved in the conversion of (S)-norcoclaurine into (S)-reticuline was once thought to proceed via a nonspecific phenol oxidase (30). However, a P450-dependent monooxygenase (CYP80B1) was shown to have a lower Km for (S)-N-methylcoclaurine than the REGULATION OF ALKALOID BIOSYNTHESIS 3 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: 40OMT, 30-hydroxy-N-methylcoclaurine 40-O-methyltransferase; 6OMT, norcoclaurine 6-O-methyltransferase; 7OMT, reticuline 7-O-methyltransferase; BBE, berberine bridge enzyme; CFS, cheilanthifoline synthase; CNMT, coclaurine N-methyltransf- erase; COR, codeinone reductase; CYP719A1, canadine synthase; CYP80A1, berbamunine synthase; CYP80B1, N-methylcoclaurine 30-hydroxylase; DBOX, dihydrobenzophenanthri- dine oxidase; DRR, 1,2-dehydroreticuline reductase; DRS, 1,2-dehydroreticuline synthase; MSH, N-methylstylopine 14-hydroxylase;