3. Introduction to Secondary Metabolism and the Biosynthesis of Natural Products

1. 3. Introduction to Secondary Metabolism and the Biosynthesis of Natural Products RA Macahig FM Dayrit

2. Introduction Metabolism: (Gr. metabole = change) the totality of the chemical changes in living cells which involves the buildup and breakdown of chemical compounds. Primary metabolism: biosynthesis, utilization and breakdown of the essential compounds and structural elements of the living organism, such as: sugars and polysaccharides; amino acids, peptides and proteins (including enzymes); fatty acids; and nucleotides. The starting materials are CO2, H2O and NH3. All organisms possess similar primary metabolic pathways and use similar primary metabolites. 3. Secondary metabolites and Biosynthesis (Dayrit)

3. Introduction Secondary metabolism: refers to the biosynthesis, utilization and breakdown of smaller organic compounds found in the cell. These compounds, called secondary metabolites, arise from a set of intermediate building blocks : acetyl coenzyme A (acetyl-CoA), mevalonic acid (MVA) and methyl erythritol phosphate (MEP), shikimic acid, and the amino acids phenylalanine/tyrosine, tryptophan, ornithine and lysine. 3. Secondary metabolites and Biosynthesis (Dayrit)

4. Introduction • Relationship between primary and secondary metabolism: • The processes and products of primary metabolism are similar in most organisms, while those of secondary metabolism are more specific. • In plants, primary metabolism is made up of photosynthesis, respiration, etc., using CO2, H2O, and NH3 as starting materials, and forming products such as glucose, amino acids, nucleic acids. These are similar among different species. • In secondary metabolism, the biosynthetic steps, substrates and products are characteristic of families and species. Species which are taxonomically close display greater similarities (and metabolites); those which are distant have greater differences. 3. Secondary metabolites and Biosynthesis (Dayrit)

5. Introduction Biogenesis: overview of the origin of compounds starting from the set of intermediate building blocks: acetyl-CoA, MVA and MEP, shikimic acid, and the amino acids phenylalanine and tyrosine, tryptophan, ornithine and lysine. Biosynthesis: detailed study of the step-wise formation of secondary metabolites. At more detailed levels, the specific enzymes, genes and signals are also identified. 3. Secondary metabolites and Biosynthesis (Dayrit)

6. * * Overview of Secondary Metabolism * Metabolites found in higher organisms only * 3. Secondary metabolites and Biosynthesis (Dayrit)

7. Metabolite linkage map representing primary and secondary plant metabolism in opium poppy. The circles associated with each metabolite indicate whether the metabolite was detected (), not detected () or masked (). (Zulak et al. BMC Plant Biology 2008 8:5; www.biomedcentral.com)

8. Biogenetic classification of natural products. 3. Secondary metabolites and Biosynthesis (Dayrit)

9. The basic biogenetic and structural groups: Acetogenins 3. Secondary metabolites and Biosynthesis (Dayrit)

10. methyl erthritol phosphate The basic biogenetic and structural groups:Terpenoids 3. Secondary metabolites and Biosynthesis (Dayrit)

11. The basic biogenetic and structural groups: Shikimates 3. Secondary metabolites and Biosynthesis (Dayrit)

12. The basic biogenetic and structural groups: Alkaloids 3. Secondary metabolites and Biosynthesis (Dayrit)

13. The following cytotoxic anthraquinone derivative was recently isolated from the stem bark of Goniothalamus marcanii Craib. Propose its biogenetic origin. Highlight the appropriate atoms in the molecule. Exercise Propose its biogenetic origin of the following alkaloid. Highlight the appropriate atoms in the molecule.

14. From Methyl methionine From Acyl-CoA From Shikimate From Methyl methionine The following cytotoxic anthraquinone derivative was recently isolated from the stem bark of Goniothalamus marcanii Craib. Propose its biogenetic origin. Highlight the appropriate atoms in the molecule. Exercises 2 & Answers 7 AcylCoA’s + 2 methyl methionines Propose the biogenetic origin of the following alkaloid. Highlight the appropriate atoms in the molecule. 2 Phenylalanines/ Tyrosines + 2 methyl methionines Chemistry of Natural Products (Dayrit)

15. Phylogenetics and natural products • Prevalence of secondary metabolites in various organisms: • Bacteria and Fungi: Fats & lipids, Acetogenins, Terpenes • Plants: +Phenylpropanoids, +Alkaloids Variations of secondary metabolism exist in various organisms. For example, recently a second pathway in the biosynthesis of terpenes in plants was discovered. The first pathway is the better-known mevalonic acid (MVA) pathway; the second pathway is the methyl erythritol phosphate (MEP) pathway which operates in the chloroplast. Many of the early biosynthetic studies were conducted using bacteria, in particular E. coli. It is possible that processes in higher organisms differ, and that revisions may appear in the future. 3. Secondary metabolites and Biosynthesis (Dayrit)

16. Phylogenetics and natural products: Evolution of terpene biosynthesis in plants 3. Secondary metabolites and Biosynthesis (Dayrit)

17. Evolution of secondary metabolism in higher plants (http://www.uk.plbio.kvl.dk/plbio/students-projects/evolution-sec-metaboites.pdf) • Cytochromes P450 and family 1 glycosyltransferases are key enzymes in biosynthesis of secondary metabolites found in higher plants. Genomic and cDNA sequencing programs of a number of model plants have unravelled a wealth of information on genes and genomes giving better understanding of evolution in terrestrial plants. • Deduced sequences of genes can be used in the analysis of phylogenetic trees to obtain their evolutionary relationship. 3. Secondary metabolites and Biosynthesis (Dayrit)

18. Introduction to Biosynthesis This section will focus on the chemical transformations of biosynthesis. It will also survey the enzymes which are responsible for these transformations. Natural products are unparalleled in the diversity and complexity of chemical structures. Despite the complexity of natural products, it should be emphasized that biosynthesis proceeds by discrete chemically reasonablesteps. That is, no matter how complicated a natural product compound is, one can rationalize its biosynthesis using a series of simple chemical transformations,. 3. Secondary metabolites and Biosynthesis (Dayrit)

19. Why study the biosynthetic pathway? • The determination of the biosynthetic pathway enables us to understand the relationships and dynamic flow of the compounds that are present in a living cell. • The objective of the study of a biochemical sequence is to be able to identify the “intermediates” and the “product”. However, there are cases when this is not so obvious. During the chemical extraction process, we obtain many of these compounds and the problem is to determine the sequence of their formation. • An understanding of a biosynthetic sequence can help us identify the enzymes and genes, understand the relationships among different organisms (such as symbiosis, plant-insect interactions, etc). An understanding of biosynthesis is part of a complete understanding of plant biology, ecology and biodiversity. 3. Secondary metabolites and Biosynthesis (Dayrit)

20. An understanding of biosynthesis is very useful! • It enables us to classify the diversity and complexity of natural products structures. • It reveals the functional relationships among natural products in a dynamic context. • It provides essential information which enables us to control or manipulate the formation of desired metabolites. • It opens up possible directions in biotechnology and molecular biology through the study of enzymes (proteomics) and genomics: • Genomics + Proteomics + Biosynthesis = Metabolonomics 3. Secondary metabolites and Biosynthesis (Dayrit)

21. Some types of biosynthetic pathways: 3. Secondary metabolites and Biosynthesis (Dayrit)

22. Some comments on biosynthetic pathways: • A compound is an obligatory intermediate if its formation is required for the biosynthetic process to continue and there are no alternative pathways. Such is the case for the compounds in a linear pathway. In comparison, a metabolic grid provides many alternative routes to the product. • Although compounds are usually transformed from simple structures to more complex ones, this is not always the case. 3. Secondary metabolites and Biosynthesis (Dayrit)

23. Some comments on biosynthetic pathways: • Different organisms may produce the same types of compounds through different pathways (e.g., convergent evolution), even if they are widely separated phylogenetically. • Some compounds may be produced by the same organism via more than one biosynthetic path. That is, there may be more than one path available, such as in a modified linear process or metabolic grid. • Even if the same compound is present in two different organisms, it is possible that they are formed via different pathways. This, however, is more likely for metabolites with simple structures. 3. Secondary metabolites and Biosynthesis (Dayrit)

24. Some comments on biosynthetic pathways: • The production of secondary metabolites depends on genetic and environmental factors. That is, secondary metabolites may be present in the organism in various amounts depending on the time of day or season, at particular stages of the organism’s life, or in response to certain environmental stimuli (e.g., production of defense compounds). • Because these compounds are produced by specific enzymes and precursors, it can be assumed that they are produced in specific parts or organelles of the plant. • Secondary metabolites are probably in a state of dynamic flux, being produced and broken down constantly. Some compounds, however, may be stored in specific organelles and have more constant presence. 3. Secondary metabolites and Biosynthesis (Dayrit)

25. General strategies for studying secondary metabolism: • Enzyme control. If the enzymes in the biosynthetic pathway are known or have been isolated, these enzymes can be blocked either by introducing enzyme inhibitors or by causing mutations which alter the activities of these enzymes. • Metabolite control. Many secondary metabolites are controlled by a feedback mechanism. It is reasonable to assume that there is a steady-state condition operating in the organism where the concentrations of the metabolites are maintained at some level. Effect on biosynthesis may be negative (inhibitory) or positive. 3. Secondary metabolites and Biosynthesis (Dayrit)

26. Strategies for studying secondary metabolism: Enzyme control Example: the biosynthetic sequence in a linear process using mutants or enzyme inhibitors 3. Secondary metabolites and Biosynthesis (Dayrit)

27. Strategies for studying secondary metabolism: Metabolite control 3. Secondary metabolites and Biosynthesis (Dayrit)

28. Examples of isotopically-label compounds used in biosynthetic studies: 3. Secondary metabolites and Biosynthesis (Dayrit)

29. Examples of isotopically-label compounds used in biosynthetic studies: 3. Secondary metabolites and Biosynthesis (Dayrit)

30. Examples of isotopically-label compounds used in biosynthetic studies: 3. Secondary metabolites and Biosynthesis (Dayrit)

31. Major chemical transformations in Biosynthesis • Hydrolysis • Esterification • Oxidation • Reduction • C-C Bond formation • Nucleophilic substitution • Elimination reaction • Cationic rearrangement 3. Secondary metabolites and Biosynthesis (Dayrit)

32. Major biosynthetic transformations 3. Secondary metabolites and Biosynthesis (Dayrit)

33. Major biosynthetic transformations 3. Secondary metabolites and Biosynthesis (Dayrit)

34. Major biosynthetic transformations 3. Secondary metabolites and Biosynthesis (Dayrit)

35. Major biosynthetic transformations 3. Secondary metabolites and Biosynthesis (Dayrit)

36. Major biosynthetic transformations 3. Secondary metabolites and Biosynthesis (Dayrit)

37. Enzymes in biosynthesis Most of the biosynthetic reactions are mediated by specific enzymes. Enzymes have five fundamental properties: • 1. increase in reaction rate - enzymes are catalysts which increase the forward and reverse rates of a chemical step. • 2. kinetic control - Enzymes are subject to various types of control, such as pH and feedback. • 3. chemoselectivity - Enzymes can distinguish functional groups. For example, in an oxidation reaction: C-H  C-OH, chemoselectivity allows the differentiation between various types of C-H, such as primary, secondary and tertiary alkyl, olefinic and aromatic positions. 3. Secondary metabolites and Biosynthesis (Dayrit)

38. Enzymes in biosynthesis • 4. regioselectivity - Regioselectivity is the ability of select only one site of reaction from a number of possibilities of the same functional group. For example, in a long chain saturated fatty acid, the initial site of dehydrogenation is typically 9,10. In a sugar, or a compound with many -OH groups, the position of methylation is specific. • 5. stereoselectivity - This refers to the chiral recognition of substrates (compare with chemoselectivity). 3. Secondary metabolites and Biosynthesis (Dayrit)

39. Stereoselectivity in biosynthesis • Classification of stereoselectivity: • Enantioselective - The reactants are enantiomeric and the enzyme reacts with only one enantiomer. • Prochiral - The carbon reaction center, CH2(R1)(R2), is not chiral, but becomes chiral with substitution of one of the hydrogens. In the case of a ketone, (R1)(R2)C=O, where R1R2, reduction of the carbonyl to an alcohol produces a chiral center at the carbon. 3. Secondary metabolites and Biosynthesis (Dayrit)

40. Control of enzyme activity • An organism must be able to regulate its enzymes so that it can coordinate its many biosynthetic activities and respond to its environment. It is reasonable to assume that the organism derives an advantage or fulfills a need when it biosynthesizes secondary metabolites. Therefore, careful control of their biosynthesis is an important ability. • There are two major types of control of biosynthesis: • inhibition of a specific enzyme by one of the metabolites (protein inhibition); and • regulation by induction or repression of gene expression. 3. Secondary metabolites and Biosynthesis (Dayrit)

41. Inhibition of enzyme activity • Feedback inhibition is one common mode of biosynthetic regulation in which the changing concentration of a product attenuates (decreases) the activity of an enzyme. • Allosteric control (Greek: allos, other + stereos, space or solid) occurs when the binding of the substrate is selectively increased or decreased by the binding of another species at a different (allosteric) site on the enzyme. 3. Secondary metabolites and Biosynthesis (Dayrit)

42. Types of feedback control of biosynthesis. • Simple mass action: In a reversible process, if the ratio of the concentrations of products over those of reactants, [P]/[R], is not equal to the equilibrium constant, K, then the equilibrium will shift accordingly. • Reversible competitive inhibition of the enzyme by the product: In this case, the product slows down its own formation by inhibition of the enzyme. • Product or reactant interacts with the DNA or RNA to induce or repress the synthesis of the enzymes which are responsible for the biosynthesis. 3. Secondary metabolites and Biosynthesis (Dayrit)

43. Some types of control of biosynthetic activity through the action of metabolites on enzymes. 3. Secondary metabolites and Biosynthesis (Dayrit)

44. Schematic representation of the mechanisms for inducing or repressing gene function. 3. Secondary metabolites and Biosynthesis (Dayrit)

45. Enzyme classification (EC) system 3. Secondary metabolites and Biosynthesis (Dayrit)

46. The IUB number and classification of enzymes 3. Secondary metabolites and Biosynthesis (Dayrit)

47. The four major types of biological oxidation reactions catalyzed by oxidoreductases 3. Secondary metabolites and Biosynthesis (Dayrit)

48. The four major types of biological oxidation reactions catalyzed by oxidoreductases 3. Secondary metabolites and Biosynthesis (Dayrit)

49. Elimination and rearrangement reactions following oxidation 3. Secondary metabolites and Biosynthesis (Dayrit)

50. Elimination and rearrangement reactions following oxidation 3. Secondary metabolites and Biosynthesis (Dayrit)