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Three of the most studied pathways, for economic and pharmacological reasons, are those involved in the production of codeine and morphine in Papaver somniferum, capsaicin and other capsaicinoids in Capsicum annuum and compounds such as rosmarinic acid from Lamiaceae herbs 15,200,201_{. These and} many other secondary metabolite pathways involve only a small collection of gene families that are used to create and modify a key precursor sometimes referred to as a scaffold(see tables 4.1, 4.2 and 4.3 for details of enzymes linked to secondary metabolites in poppies, Capsicum and Lamiaceae)202_{. The} generation of these precursors involves the use of primary metabolite substrates as building blocks for several precursors generated through a small set of enzymatic reactions 202_{. These precursor, such as strictosidine for terpene} indole alkaloids and norcoclaurine for BIAs, are then modified to create the large and diverse number of metabolites seen within these secondary metabolite families 110,203,204_.

Modification of the key precursor can occur either via redox chemistry such as oxidation or through group transfers such as alkylation, acylation and glycosylation 202_{. The enzymes involved in these modifications can often be} related but with different substrate specificity, or can be part of a pathway involving several different enzyme classes 202_.

The following discussion focuses in on two classes of enzymes that are involved in alkaloid production in several plant systems and could be suggested for the galanthamine pathway, namely P450 monooxidases and Pictet-Spenglerases 17,203,205,206_{. These will be discussed in relation to their involvement in secondary} metabolism and the pathways chosen for investigation to create a database of predicted gene homologs.

4.1.3.1 P450 subfamilies linked to secondary metabolism

Cytochrome P450s are a class of haem enzymes named for their maximum optical absorbance at 450 nm (in a reduced state complex with carbon monoxide) 207_{. They are found in numerous plant organelles including the} endoplasmic reticulum, mitochondria, plastids and Golgi bodies 208_{. The first} plant P450 was identified in 1969 in cotton and the first to be sequenced was CYP71A1 from avocado 209,210_{. Amino acid similarity is used in the classification} of P450s. If the similarity between two is over 40% they belong to the same family. Within a family, a similarity above 55% classifies the proteins within the same subfamily 208_.

P450s are involved in many processes in both primary and secondary metabolism and are predicted to account for up to 1% of plant genome annotations. For example there are 246 P450s annotated in Arabidopsis, 356 in rice, 312 in poplar and 457 in grape 211_{. Within secondary metabolism P450s} catalyse a variety of monooxygenation and hydroxylation reactions. They also catalyse four types of unusual reactions, specifically methylenedeoxy bridge formation, phenol coupling, oxidative rearrangement of carbon skeletons and oxidative C-C bond cleavage 211_{. The first two types of reaction are now} discussed in greater detail due to their involvement in alkaloid biosynthesis 211,212_.

4.1.3.2 Methylenedeoxy-bridge formation

This reaction involves the formation of a bridge via oxidative cyclisation of an ortho-hydroxymethoxy-substituted aromatic ring 211_{. Within isoquinoline} alkaloid biosynthesis these reactions are catalysed by P450s. These reactions are deemed unusual since they do not involve the stereotypical hydroxylation step of inserting an oxygen molecule 213_.

Figure 4-1 A simplified methylenedeoxy-bridge formation.

The clan responsible for bridge formation in isoquinoline alkaloid biosynthesis is CYP719A in a reaction where a hemiacetal intermediate of formaldehyde (formed via a P450-dependent hydroxylation of a methoxy group) is cyclized via an ionic mechanism to produce the methylenedeoxy-bridge 214-216_{. These} enzymes are all substrate specific and so several different CYP719As are responsible for similar reactions throughout alkaloid biosynthesis. CYP719A1 converts tetrahydrocolumbamine to (S)- tetrahydroberberine in canadine synthesis in Japanese goldthread (Coptis trifolia) 217_{. In Eschscholzia californica} two CYP719As are involved in stylopine synthesis but show differing substrate specificity. CPY719A2 has high affinity for (R, S)-cheilanthifoline alone, while CYP719A3 has affinity for three similar substrates, (R, S)-cheilanthifoline, (S)- scoulerine and (S)-tetrahydrocolumbamine 218_.

4.1.3.3 Phenol coupling

The final step in the biosynthesis of galanthamine is an intramolecular para- ortho phenol coupling reaction. Reactions like this are seen in numerous alkaloid biosynthesis pathways and are often catalysed by CYP80 or CYP719 enzymes 205,212_{. Intramolecular C-C phenol coupling is required in BIA synthesis} and is catalysed by both CYP80G2 and CYP719B1 depending on the substrate. In C. japonica CYP80G2 converts (S)-reticuline to (S)-corytuberine whereas in the synthesis of salutaridine as part of the morphine biosynthetic pathway in P.

somniferum, CYP719B1 converts (R)-reticuline to salutaridine 205,219_{. In contrast} both the S and R configurations of N-methylcoclaurine in bisbenzylisoquinoline alkaloid biosynthesis can be converted by the same P450, CYP80A1, catalyzing an intermolecular C-O phenol coupling reaction to form berbamunine 220_.

Both CYP719B1 and CYP80G2 show high levels of sequence similarity to other P450s but different substrate specificity and therefore catalyse different reactions (CYP719B1 shares 51% amino acid similarity with CYP719A1 and CYP80G2 shares 52% amino acid similarity with CYP80A1)205,219_{. It is therefore} important to examine function as well as sequence similarity when predicting possible homologs in daffodils and this is particularly important in P450s. The fact that some P450s are highly substrate specific and others have broader substrate specificity makes it difficult to predict which family or subfamily will be involved in galanthamine biosynthesis and several will therefore be included in the predicted database.

4.1.3.4 PSRs role in secondary metabolism

Pictet Spengler reactions are condensation reactions employed in the synthesis of alkaloids, named after Amé Pictet and Theodor Spengler who discovered them 221_{. They synthesised the alkaloid 1,2,3,4, tetrahydroisoquinoline via a} cyclo-addition reaction between β-phenylethylamine and formaldehyde 221_{. The} first plant enzyme that catalysed this type of reaction was found in Catharanthus roseus. The Pictet Spenglerase (PSR) strictosidine synthase is involved in the biosynthesis of strictosidine, the key precursor of monoterpenoid indole alkaloids 222,223_{. Strictosidine synthase was first purified} in 1979 and since then has been used as a biomimetric synthase in the synthesis of novel alkaloids 206_{. Although strictosidine synthase is a member of the 6-} bladed β propeller protein family, not all PSRs are from this protein family 203,206,224_.

Figure 4-2 STR catalyzing a PSR reaction.(adapted from Stockigt et al., 2011) 206.

PSRs are an example where several gene/enzyme families catalyse the same type of reaction. In BIA biosynthesis a similar reaction is carried out via a Bet v1/PR10 family protein known as norcoclaurine synthase (NCS) 204_{. NCS}

catalyses an asymmetric PS condensation of dopamine and 4-

hydroxyphenylacetaldehyde to synthesize (S)-norcoclaurine in the first dedicated step of morphine and codeine biosynthesis 18_{. NCS has been isolated} from several plants including P. somniferum and Thalictrum flavum 203,204_{. Work} has also been carried out to see if PSRs have similar ancestry or share sequence similarity. NCS and do not share any homology and so it is predicted that they evolved to carry out similar reactions from different ancestral proteins 225_.

The study of both P450s and PSRs involved in secondary metabolism shows the need for analysis of sequence similarity, gene families, enzymatic reaction types and metabolite and transcript levels in the search for putative genes. In the search for possible genes involved in galanthamine production, all of the above methods will be utilized.