Redesign of a dioxygenase in morphine biosynthesis

Opium poppy (Papaver somniferum) produces medicinally important benzylisoquinoline alkaloids, including the analgesics codeine and morphine, in the morphinan pathway. We aligned three dioxygenases that were recently discovered in P. somniferum and subsequently identified the nonconserved regions. Two of these enzymes, codeine O-demethylase (PsCODM) and thebaine O-demethylase (PsT6ODM), are known to facilitate regioselective O-demethylation in morphinan biosynthesis. We systematically swapped the residues that were nonconserved between the PsCODM and PsT6ODM sequences to generate 16 mutant PsCODM proteins that could be overexpressed in Escherichia coli. While wild-type PsCODM can demethylate both codeine and thebaine, one engineered PsCODM mutant selectively demethylates codeine. Use of this reengineered enzyme in the reconstitution of morphine biosynthesis could selectively disable a redundant pathway branch and therefore impact the yields of the downstream products codeine and morphine in subsequent metabolic engineering efforts.     

Rapid identification of enzyme variants for reengineered alkaloid biosynthesis in periwinkle

Monoterpene indole alkaloids from Catharanthus roseus (Madagascar periwinkle), such as the anticancer agents vinblastine and vincristine, have important pharmacological activities. Metabolic engineering of alkaloid biosynthesis can provide an efficient and environmentally friendly route to analogs of these synthetically challenging and pharmaceutically valuable natural products. However, the narrow substrate scope of strictosidine synthase, the enzyme at the entry point of the pathway, limits a pathway engineering approach. We demonstrate that with a different expression system and screening method it is possible to rapidly identify strictosidine synthase variants that accept tryptamine analogs not turned over by the wild-type enzyme. The variants are used in stereoselective synthesis of beta-carboline analogs and are assessed for biosynthetic competence within the terpene indole alkaloid pathway. These results present an opportunity to explore metabolic engineering of "unnatural" product production in the plant periwinkle.     

Foundations for directed alkaloid biosynthesis

In this issue of Chemistry & Biology, Bernhardt and coworkers [1] assay the functional plasticity of strictosidine synthase, a gateway enzyme in the biosynthetic pathways of monoterpene indole alklaloids, and the downstream operability of the products of strictosidine synthase variants in the larger context of the plant biosynthetic pathways.     

The carbon-skeleton rearrangement in tropane alkaloid biosynthesis

High-level quantum chemistry calculations have been performed to examine the carbon-skeleton rearrangement of the tropane alkaloid littorine to hyoscyamine. Two pathways involving radical and carbocation intermediates have been investigated in this regard, namely, stepwise (or fragmentation-recombination) and concerted. The fragmentation products are calculated to be of high energy for both the radical- and carbocation-based mechanisms (136.3 and 170.9 kJ mol(-1), respectively). Similarly, the rearrangement barrier for the radical-based concerted pathway is calculated to be quite high (135.6 kJ mol(-1)). In contrast, the carbocation-based concerted pathway is found to be associated with a relatively low barrier (47.4 kJ mol(-1)). The ionization energy of the substrate-derived radical 3a is calculated to be 7.01 eV, suggesting that its oxidation to generate the substrate-derived carbocation 3b ought to be facile. In an attempt to investigate how an enzyme might modulate the rearrangement barriers, the separate and combined influences of partially protonating the migrating group and partially deprotonating the spectator OH group of the substrate were investigated. Such interactions can lead to significant reductions in the rearrangement barrier for both the radical- and carbocation-based concerted pathways, although the carbocation pathway continues to have significantly lower energy requirements. Also, the relatively high (gas-phase) acidity of the OH group of the product-related carbocation 4b indicates that the direct formation of hyoscyamine aldehyde (6) is a highly exothermic process. Although we would not wish to rule out alternative possibilities, our calculations suggest that a concerted rearrangement mechanism involving carbocations constitutes a viable low-energy pathway for the carbon-skeleton rearrangement in tropane alkaloid biosynthesis.     

The impact of structural biology on alkaloid biosynthesis research

Covering: 1997 to 2012In the recent past, macromolecular crystallography has gone through substantial methodological and technological development. The purpose of this review is to provide a general overview of structural biology and its impact on enzyme structure/function analysis and illustrate how it is modifying the focus of research relevant to alkaloid biosynthesis.     

Discovery and efficient synthesis of a biologically active alkaloid inspired by thiostrepton biosynthesis

Thiostrepton, a natural peptide macrocycle, is of great interest due to its structural complexity and numerous biological activities, including anti-bacterial, anti-tumor, and anti-plasmodial activities. The quinaldic acid (QA) moiety-containing side ring (loop 2) was proven to play an important role in carrying out these functions. Previously, we proposed biosynthetic logic for thiostrepton loop 2 and demonstrated the formation mechanism of QA. Herein, we report the discovery and efficient synthesis of a biologically active alkaloid, that is, a key intermediate involved in the thiostrepton biosynthetic pathway. A chemo-enzymatic method was performed to synthesize the molecule, and a series of analogs were prepared for bioassays, which included the examination of anti-bacterial and anti-tumor activities.     

Directed biosynthesis of alkaloid analogs in the medicinal plant Catharanthus roseus

Terpene indole alkaloids are plant natural products with diverse structures and biological activities. A highly branched biosynthetic pathway is responsible for the production of approximately 130 different alkaloids in Madagascar periwinkle (C. roseus) from a common biosynthetic intermediate derived from tryptamine. Although numerous biosynthetic pathways can incorporate unnatural starting materials to yield novel natural products, it was not clear how efficiently the complex, eukaryotic TIA pathway could utilize unnatural substrates to make new alkaloids. This work demonstrates that the TIA biosynthetic machinery can be used to produce novel alkaloid structures and also highlights the potential of this pathway for future metabolic engineering efforts.     

Enzymatic study of the late stages of protoberberine alkaloid biosynthesis

A specific methyltransferase which converts tetrahydrocolumbamine to tetrahydropalmatine, and oxidase activity which converts tetrahydroberberine, tetrahydrocolumbamine and tetrahydropalmatine, but not tetrahydrojatrorrhizine, to their quaternary counterparts are demonstrated.     

Mechanism-based enzyme inactivators of phytosterol biosynthesis

Current progress on the mechanism and substrate recognition by sterol methyl transferase (SMT), the role of mechanism-based inactivators, other inhibitors of SMT action to probe catalysis and phytosterol synthesis is reported. SMT is a membrane-bound enzyme which catalyzes the coupled C-methylation-deprotonation reaction of sterol acceptor molecules generating the 24-alkyl sterol side chains of fungal ergosterol and plant sitosterol. This C-methylation step can be rate-limiting in the post-lanosterol (fungal) or post-cycloartenol (plant) pathways. A series of sterol analogs designed to impair SMT activity irreversibly have provided deep insight into the C-methylation reaction and topography of the SMT active site and as reviewed provide leads for the development of antifungal agents.     

Characteristics of vellosimine reductase,a specific enzyme involved in the biosynthesis of the rauwolfia alkaloid sarpagine

A plant enzyme - vellosimine reductase - has been isolated from $\text{Rauwolfia}$ cell suspension cultures. This new enzyme has been-purified (110-fold) and characterized. The reductase is a specific enzyme of the sarpagine pathway catalyzing the NADPH dependent conversion of vellosimine into 10-deoxysarpagine. The latter alkaloid is the immediate biogenetic precursor of sarpagine as shown by its high $\text{in}$$\text{vivo}$ incorporation rate (86%) into sarpagine.     

Studies on the synthesis and biosynthesis of the fungal alkaloid necatorone

An alternative pathway for the biosynthesis of the fungal alkaloid necatorone has been studied using fluorine-labeled 3-(2-carboxyphenylamino)-l-tyrosine. Although no incorporation of this compound could be detected in feeding experiments with young specimens of Lactarius necator, an analogous 3-aminotyrosine derivative could be converted synthetically into the oxopyridoacridine core structure of necatorone. In experiments aimed at the synthesis of aaptamine-type alkaloids, an unprecedented cyclization of a 3-aminotyrosine-methyl propiolate adduct to a methyl isoquinoline-3-carboxylate was observed. A mechanism is proposed, in which C3 of the propiolate delivers C1 of the isoquinoline nucleus.     

Cucurbitadienol synthase, the first committed enzyme for cucurbitacin biosynthesis, is a distinct enzyme from cycloartenol synthase for phytosterol biosynthesis

Three oxidosqualene cyclase (OSC) cDNAs (CPX, CPQ, CPR) were cloned from seedlings of Cucurbita pepo by homology based PCR method. Their open reading frames were expressed in lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. Analyses of in vitro enzyme activities and in vivo accumulated products in the transformants demonstrated that CPQ and CPX encode cucurbitadienol and cycloartenol synthases, respectively. These results indicated the presence of distinct OSCs for cycloartenol and cucurbitadienol synthesis in this plant.