Enzymatic oxidations in the biosynthesis of complex alkaloids

Abstract

SummaryThe biosynthesis of complex alkaloids in plants involves enzymes that, due to high substrate specificity, appear to have evolved solely for a role in secondary metabolism. At least one class of these enzymes, the oxidoreductases, catalyze transformations that are in some cases difficult to chemically mimick with an equivalent stereo-or regiospecificity and yield. Oxidoreductases are frequently catalyzing reactions that result in the formation of parent ring systems, thereby determining the class of alkaloid that a plant will produce. The oxidoreductases of alkaloid formation are a potential target for the biotechnological exploitation of medicinal plants in that they could be used for biomimetic syntheses of alkaloids. Analyzing the molecular genetics of alkaloid biosynthetic oxidations is requisite to eventual commercial application of these enzymes. To this end, a wealth of knowledge has been gained on the biochemistry of select monoterpenoid indole and isoquinoline biosynthetic pathways, and in recent years this has been complemented by molecular genetic analyses. As the nucleotide sequences of the oxidases of alkaloid synthesis become known, consensus sequences specific to select classes of enzymes can be identified. These consensus sequences will potentially facilitate the direct cloning of alkaloid biosynthetic genes without the need to purify the native enzyme for partial amino acid sequence determination or for antibody production prior to cDNA isolation. The current state of our knowledge of the biochemistry and molecular genetics of oxidases involved in alkaloid biosynthesis is reviewed herein.

Characterization of a Flavoprotein Oxidase from Opium Poppy Catalyzing the Final Steps in Sanguinarine and Papaverine Biosynthesis

“…S1). A phylogenetic tree based on an alignment of protein sequences corresponding to functionally characterized plant flavoproteins shows high bootstrap support for a monophyletic clade containing BBE homologues from E. californica (36), barberry (B. wilsoniae) (37), and opium poppy (38) (Fig. 2).…”

Berberine bridge enzyme, a key branch-point enzyme in benzylisoquinoline alkaloid biosynthesis, contains a vacuolar sorting determinant

“…Each construct encoded the indicated region of BBE fused to either GUS or GFP, and was¯anked by the bbe1 promoter and the nos transcription terminator. N-terminal amino acids of BBE from opium poppy (PsBBE; Facchini et al 1996) are shown aligned to those from Eschscholzia californica (EcBBE; Dittrich and Kutchan 1991) and Berberis stolonifera (BsBBE; Chou and Kutchan 1998). The arrowhead indicates the signal-peptide cleavage site identi®ed in E. californica BBE Forty-eight hours after the bombardment of opium poppy cells with pBBE::GFP, GFP was visible in the nucleoplasm and cytosol of all¯uorescent cells (Fig.…”

Characterization of proteins in latex of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresis and microsequencing

“…Protein S24 was shown to be homologous to desacetoxyvindoline-4-hydroxylase, an enzyme involved in the biosynthesis of the indole alkaloid vindoline in Catharanthus roseus [55]. Its detection in the latex of P. somniferum raises the question which role this 2-oxoglutaratedependent dioxygenase plays in the isoquinoline alkaloid biosynthesis.…”