Strictosidine glucosidase (SGD) from Catharanthus roseus catalyzes the deglycosylation of strictosidine, an intermediate from which thousands of monoterpene indole alkaloids are derived. The steady state kinetics of SGD with a variety of strictosidine analogs revealed the substrate preferences of this enzyme at two key positions of the strictosidine substrate. Additionally, SGD from C. roseus turns over both strictosidine and its stereoisomer vincoside, indicating that although this enzyme prefers the naturally occurring diastereomer, the enzyme is not completely diastereoselective. The implications of the substrate specificity of SGD in metabolic engineering efforts of C. roseus are highlighted.Monoterpene indole alkaloids (MIA) are a large class of pharmaceutically valuable and structurally complex natural products. 1 Directed biosynthesis studies have shown that the MIA pathway can produce a variety of "unnatural" alkaloids by utilizing non-natural substrate analogs. 2 This inherent flexibility suggests that MIA biosynthesis could provide a robust platform for metabolic engineering. However, not all substrate analogs are likely to be incorporated into the pathway with equal efficiency. If substrate specificity of individual biosynthetic enzymes correlates with rate limiting steps in vivo, then enzymes having a low catalytic efficiency for a non-natural substrate could be reengineered to improve turnover of the analog. 3 Therefore, evaluation of enzyme substrate specificity is critical for biosynthetic engineering efforts. In MIA biosynthesis, the central biosynthetic precursor strictosidine 1 is deglycosylated by strictosidine glucosidase (SGD) to yield a reactive intermediate that rearranges to form the wide variety of MIA (Scheme 1). 4 Here we evaluate the substrate specificity of SGD from Catharanthus roseus with a variety of strictosidine analogs to determine whether SGD could act as a bottleneck in the production of novel alkaloids from unnatural strictosidine analogs.SGD was assayed with strictosidine analogs 2-9 (Table 1). 5-8 An HPLC assay was used to monitor both strictosidine disappearance and deglycosylated product formation. Since all kinetic data appeared to fit a sigmoidal rather than a Michaelis-Menten curve, kinetic constants were obtained from a sigmoidal fit to the data ( Figure 1). 9,10 SGD from C. roseus has been reported to form aggregates consisting of 4 to 12 monomers; 4a although a sigmoidal fit has not been previously reported for C. roseus SGD, the oligomeric state of the enzyme is compatible with the cooperative mechanism suggested by the sigmoidal curve.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the c...

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Biosynthetic pathways can be hijacked to yield novel compounds by introduction of novel starting materials. Here we have altered tryptamine, which serves as the starting substrate for a variety of alkaloid biosynthetic pathways, by replacing the indole with one of four aza-indole isomers. We show that two aza-tryptamine substrates can be successfully incorporated into the products of the monoterpene indole alkaloid pathway in Catharanthus roseus. Use of unnatural heterocycles in precursor directed biosynthesis, in both microbial and plant natural product pathways, has not been widely demonstrated, and successful incorporation of starting substrate analogs containing the aza-indole functionality has not been previously reported. This work serves as a starting point to explore fermentation of aza-alkaloids from other tryptophan and tryptamine derived natural product pathways.

Total synthesis of glycosylated seco-iridoid stereoisomers allows the identification and bypassing of the stereoselectivity of early steps in monoterpene indole alkaloid biosynthesis.Stereochemistry often determines how a natural product interacts with biological systems. For example, different stereosisomers can have different pharmacological profiles. 1 The biosynthetic enzymes that form natural products typically allow the production of only one stereoisomer, so fermentation of products with alternate stereochemistry must therefore bypass the stereochemical restrictions of biosynthetic pathway enzymes. Using synthetic seco-iridoid and strictosidine starting materials we show that the heteroyohimbine branch of monoterpene indole alkaloid (MIA) biosynthesis in the medicinal plant Catharanthus roseus 2,3 has a surprisingly broad tolerance for stereochemical perturbations in vitro.Strictosidine synthase catalyzes the first step in monoterpene indole alkaloid biosynthesis: an asymmetric Pictet-Spengler reaction between tryptamine 1 and secologanin 2 to yield strictosidine 3a (Scheme 1). 4,5 In the second step of this alkaloid pathway, 3a is deglucosylated by strictosidine-β-D-glucosidase. 6,7 This results in the formation of a hemiacetal that rearranges into a mixture of products that is channeled into one of several pathway branches. The enzymes responsible for these branching reactions are unknown at the genetic and biochemical levels. However, entry into the heteroyohimbine class of alkaloids (5a-c) is likely controlled by one or several NADPH-dependent reductases (Scheme 1). 8,9 Different heteroyohimbine alkaloid stereoisomers have different pharmacological activities. The heteroyohimbine alkaloid ajmalicine (raubasine) 5a acts as a smooth muscle relaxant and as an α1 anti-adrenergic, 10-12 while tetrahydroalstonine 5b acts as an α2 anti-adrenergic. 12 The stereogenic centers of strictosidine 3a, the central intermediate for all monoterpene indole alkaloids, are either derived from the densely functionalized secologanin dihydropyran (C-15, C-20, and C-21) or the prochiral aldehyde carbon that is converted to the C-3 stereogenic center in strictosidine (Scheme 1). We recently reported that strictosidine glucosidase promotes not only deglucosylation of strictosidine 3a, but also deglucosylation of vincoside 3b (the 3-(R) diastereomer of 3a). 13 We report here that strictosidine glucosidase has an 80-fold higher specificity constant (k cat /K M ) for 3a compared to 3b ( (Table 1). In the crystal structure of SGD from the closely related Rauvolfia serpentina homolog (PDB: 2FJ6), 14 the C-3 carbon atom of strictosidine 3a is located near the surface of the protein. We speculate that the orientation of the glucose moiety, which is in the interior of the glucosidase, remains unchanged during turnover of vincoside 3b, while the binding pocket for the more distal regions of the substrate, including the C-3 carbon, can accommodate 3b.We asked whether strictosidine glucosidase can catalyze the conversion of alt...