The enzymology of these enzymes has been well-studied and the reader can refer to other reviews for more information.Here we will briefly summarize a few enzyme-catalyzed or enzyme-mediated reactions that will be found throughout the review. The aromatic amino acids L-tryptophan , L-tyrosine and to a less extent, L-phenylalanine, are commonly used precursors for alkaloid natural product biosynthesis. For example, the indole ring in L-tryptophan is preserved in compounds such as psilocybin and ibogaine; while the parahydroxybenzene side chain in L-tyrosine can be found in mescaline and morphine. The terminal amine-containing L-lysine and L-ornithine are also used as precursors. Relevant to this review, the four-carbon side chain of L-ornithine is required for the formation of pyrrolidines and tropanes. The first step in the utilization of these amino acids for alkaloid biosynthesis is decarboxylation to give the corresponding primary amines, although in lysergic acid biosynthesis L-tryptophan is used without decarboxylation. The decarboxylation products of L-tryptophan, L-tyrosine and L-ornithine are tryptamine , tyramine , and putrescine , respectively . In the case of tyramine , hydroxylation of one of the meta positions in the para-phenol ring gives the metabolite dopamine. Dopamine is a natural product building block, but also a neurotransmitter in mammals. The chemical logic for the early decarboxylation is straightforward: to facilitate intra- and intermolecular Mannich reactions with aldehydes and ketones using the nucleophilic amine .
This decarboxylation-Mannich two step rapidly sets up the -heterocyclic scaffold of many alkaloidal natural products. The decarboxylation reactions are catalyzed by dedicated amino acid decarboxylases. For example,plant bench indoor in the case of L-tryptophan, a tryptophan decarboxylase is involved. These enzymes typically use the PLP cofactor, as expected for many enzymes that perform Cα, Cβ and Cγ modifications on amino acids.The mechanism of the reaction is shown in Fig. 2B. The aldehyde of PLP modifies an active site lysine to form the resting aldimine in the decarboxylase active site. A transaldimination step takes place next in which the amine of the substrate amino acid attacks the aldimine and forms the amino acid–PLP aldimine. The PLP then serves as an electron sink in the enzyme-catalyzed cleavage of the Cα-COO− bond via a quinonoid species. Reprotonation of the Cα then generates the product aldimine, which can undergo another transaldimination with the active site lysine to release the product amine and regenerate the resting aldimine. Following decarboxylation of the amino acids to the corresponding primary amines, a common next step is the Mannich reaction involving the primary amine. The Mannich reaction is a two-step reaction that yields an alkylated amine.In the first step, the primary amine reacts with either an aldehyde or a ketone to form the Schiff base. The C=N double bond is then attacked by a carbon nucleophile, such as the acidic Cα of a carbonyl to form the β-amino-carbonyl product. Two examples of an intramolecular Mannich reaction can be found in the formation of the tropane unit in cocaine.Starting from putrescine , methylation of one of the primary amines gives the intermediate N-methylputrescine ; oxidation and hydrolysis of the other amine yields N-methylaminobutanal , which is in equilibrium with the cyclic N-methylpyrrolinium . Attack of the imine by the enolized 3-oxo-glutaric acid yields the adduct pyrrolidine tropane scaffold precursor .
A subsequent dehydrogenation generates a new pyrrolinium species that can be attacked with Cα of the 1,3-diketo unit in a second Mannich reaction . One variation of the Mannich reaction that is central to the biosynthesis of plant alkaloids is the Pictet-Spengler reaction involving β-arylethylamines such as tryptamine and dopamine. In the PS reaction, after the amine reacts with an aldehyde or ketone to form the Schiff base, a carbanion resonance structure of the indole in tryptamine or the parahydroxy phenol ring in dopamine can attack the imine to form the new C–C bond. This can be followed by rearrangements to form the stable tricyclic tetrahydro-β-carboline or bicyclic tetrahydroisoquinoline, respectively. The tryptamine-derived tetrahydro-β-carboline is found in harmala alkaloids and iboga alkaloids . To generate the harmala family of compounds, tryptamine is condensed with pyruvic acid , followed by attack of the imine by C3 from the indole ring to form a spirocycle, which collapses via single bond migration to complete the PS reaction .Similarly, the condensation between the aldehyde donor secologanin and tryptamine is catalyzed by a dedicated PictetSpenglerase, yielding strictosidine, the universal precursor to monoterpene indole alkaloids including ibogaine.In the biosynthesis of benzylisoquinoline alkaloids such as morphine , the PS reaction takes place between dopamine and 4- hydroxyphenylacetaldehyde , both oxidation products of tyramine , to form the key intermediate S-norcoclaurine , precursor to R-reticuline and morphine.Group transfer reactions are widely used by Nature in the biosynthesis of natural products. Functional groups that are frequently transferred from donor molecules to bio-synthetic intermediates include methyl, acetyl, small, medium and long alkyl-substituted acyl chains, isoprenyl, glucosyl, etc.
These reactions serve a multitude of purposes, including i) increasing the size and complexity of the molecules; ii) changing the lipophilicity of molecules; iii) altering the reactivity of functional groups; iv) serving as a transient chemical protection group for downstream modifications; v) acting as leaving groups in elimination reactions; and vi) changing the biological properties of the natural product. Hence, these reactions are indispensable to the structural diversity of natural products that have been isolated to date. The donor molecules, those that “carry” the groups to be transferred, are kinetically stable and thermodynamically activated: the molecules are high in energy and therefore releasing the groups is a highly exergonic reaction; yet the molecules are stable under cellular conditions and enzyme catalysis is required to overcome the kinetic barriers. We recently reviewed eight such molecules that power cellular metabolism, which include ATP, NADH, acetyl-CoA, SAM, carbamoyl phosphate, isoprenyl pyrophosphate, UDP-glucose and molecular oxygen.NADH and molecular oxygen are involved in the redox reactions and will be summarized in the next section. Among the remaining six, carbamoylphosphate is involved in nitrogen metabolism and is not directly involved in natural product biosynthesis. The remaining five, however, are frequently used group transfer donor molecules,greenhouse rolling racks and examples can be found throughout the review. ATP, the universal cellular energy currency, is the donor in the transferring of phosphate groups to nucleophilic oxygen in the presence of a phosphotransferase. This reaction is ubiquitous in primary metabolism but is quite rare in natural product biosynthesis . One such example can be found in the psilocybin pathway . Acetyltransferases catalyze the transfer of acetyl groups from the acetyl-CoA thioester to a variety of O and N nucleophiles . SAMdependent methyltransferases use S-adenosylmethionine to transfer a methyl group from the trivalent sulfonium group to C, O, N, and S nucleophiles in an SN2 type substitution reaction . This reaction can be found in the majority of bio-synthetic pathways described herein. For example, iterative N-methylation of tryptamine yields the psychoactive molecule N,N-dimethyltryptamine . UDP-glucose is an activated glucose donor in cells for the assembly of oligosaccharides and polysaccharides. UDP-glucose is thermodynamically activated but kinetically stable in the absence of glucosyltransferases.In the presence of glucosylating enzymes, UDP dissociates via cleavage of the C–O bond in an SN1 fashion to yield a C1 oxocarbonium ion, which can be attacked by incoming nucleophiles . A notable example of substrate glucosylation is in the bio-synthetic pathway of strictosidin, the precursor to ibogaine . The enzyme 7DLGT glucosylates the hemiacetal in 7-deoxyloganetic acid to give 7-deoxyloganic acid.The glucose moiety serves as a protecting group to prevent formation of the aldehyde, and remains in strictosidine . In order to transform strictosidineinto different scaffolds, a glucosidase removes the glucose moiety, unmasking the aldehyde and leading to subsequent rearrangements towards structurally diverse monoterpene indole alkaloids. The final group transfer reaction that is relevant to this review is the transfer of prenyl groups from isoprenyl pyrophosphate to different nucleophiles in small molecules. These reactions are catalyzed by a family of enzymes known as prenyltransferases.
The prenyl unit that is transferred from the pyrophosphorylated donor to the substrate can be as small, as in the five-carbon dimethylallyl , or the more elongated oligoprenyl groups such as the ten-carbon geranyl, fifteen-carbon farnesyl, etc. In the enzyme active site, the Δ2 – prenyl pyrophosphate donors can undergo C–O bond cleavage to yield the C1 carbocation, which is stabilized by delocalization of the positive charge. Attack of the carbocation by a nucleophile carbon forges the new bond and completes the prenyl transfer reaction . Electron rich aromatic rings, such as hydroxybenzenes and indoles can serve as nucleophiles to attack the allyl cation to perform in essence an electrophilic aromatic substitution. Two examples in this review illustrate this reaction. The first is the dimethylallyl tryptophan synthase in lysergic acid biosynthesis, which prenylates the C4 position in L-tryptophan to give 4-dimethylallyl-L-tryptophan .This modification introduces an olefin-containing five carbon unit into L-tryptophan, which can be further oxidized and cyclized into the hallucinogenic lysergic acid. The mechanism of this reaction has been thoroughly studied, and is likely a two-step reaction.The C3 position of the indole ring is the most nucleophilic due to resonance with the indole nitrogen lone pair. Attack on the allyl cation can occur at either C1 or C3; this attack is proposed to take place at the more stable C3 position of the allyl cation. This generates a “reverse”-prenylated product that is proposed to undergo a nonenzymatic sigmatropic Cope rearrangement to yield the “forward”-prenylated 4-DMAT. In addition to serving as the starting point for lysergic acid , indole prenylation of early pathway intermediates is commonly observed in the biosynthesis of other fungal indole alkaloids. The second notable pathway that involves prenyl transfer is in cannabinoid biosynthesis .Starting with the first intermediate in the pathway, olivetolic acid which is a resorcinol derivative, the aromatic prenyltransferase transfers the ten-carbon geranyl group from geranyl pyrophosphate to the C3 position in the ring to give cannabigerolic acid . As in the lysergic acid example, the introduced ten-carbon unit can undergo oxidative intramolecular cyclization, providing a variety of cannabinoids . Natural product bio-synthetic pathways employ powerful redox enzymes to modify the intermediates en route to the final product. The redox modification can directly modify the molecular scaffolds, or trigger rearrangement cascades, to introduce considerable structural complexities.On the reductive side, the NADH utilizing enzymes dominate as one would expect. These include ketoreductases, short-chain dehydrogenase/reductases , ene-reductases, and imine reductases, etc. The two-electron reduction of C=C, C=O or C=N bonds are initiated through the attack by a hydride equivalent from either the dihydropyridine ring of NADH or the hydroquinone form of flavin adenine dinucleotide . On the oxidative side, aerobic organisms use an assortment of enzymes and molecular oxygen as the oxidant to perform a dazzling array of chemical modifications.Both single electron and two electron manifolds are used by enzymes. These enzymes include the large family of hemedependent cytochrome P450 monooxygenases that are abundant in plants and fungi; nonheme, iron and α-ketoglutarate dependent oxygenases, copper-dependent oxidases , and flavin-dependent monooxygenases and oxidases. In two-electron oxidation of substrates catalyzed by oxidases, molecular oxygen is reduced to hydrogen peroxide. In monooxygenases where oxygen is reduced fully to water , the substrate undergoes a two-electron oxidation, while NADPH is oxidized to NADP+. Here, the substrate can incorporate one of the oxygen atoms via hydroxylation or epoxidation, or alternatively the substrate can be oxidized without incorporation of oxygen atoms. Hence, depending on the mechanism of the redox enzyme, the outcome of the reaction can be very different. This topic has been extensively reviewed in the literature, and will not be discussed in detail here. However, we will highlight two reactions to illustrate the enzymatic prowess of the P450s, a staple of the plant bio-synthetic pathways. P450 enzymes use heme as a coenzyme to bind molecular oxygen. Binding of molecular oxygen and electron transfer from the Fe and CPR leads to a hydroperoxy Fe–O–O–H species. Cleavage of the O–O bond and the loss of water generates the high valent Fe=O porphyrin cation radical, which is also referred to as Compound I. This is a highly oxidizing species that can abstract hydrogen from substrate C, O, and N atoms to generate substrate radicals, including “unactivated” sp 3 carbons. This generates the Fe– OH species also known as Compound II. Radical OH transfer to the substrate carbon radical produces the hydroxylated product in a process known as oxygen rebound.