Mining Enzyme Diversity of Transcriptome Libraries through DNA Synthesis for Benzylisoquinoline Alkaloid Pathway Optimization in Yeast.

The ever-increasing quantity of data deposited to GenBank is a valuable resource for mining new enzyme activities. Falling costs of DNA synthesis enables metabolic engineers to take advantage of this resource for identifying superior or novel enzymes for pathway optimization. Previously, we reported synthesis of the benzylisoquinoline alkaloid dihydrosanguinarine in yeast from norlaudanosoline at a molar conversion of 1.5%. Molar conversion could be improved by reduction of the side-product N-methylcheilanthifoline, a key bottleneck in dihydrosanguinarine biosynthesis. Two pathway enzymes, an N-methyltransferase and a cytochrome P450 of the CYP719A subfamily, were implicated in the synthesis of the side-product. Here, we conducted an extensive screen to identify enzyme homologues whose coexpression reduces side-product synthesis. Phylogenetic trees were generated from multiple sources of sequence data to identify a library of candidate enzymes that were purchased codon-optimized and precloned into expression vectors designed to facilitate high-throughput analysis of gene expression as well as activity assay. Simple in vivo assays were sufficient to guide the selection of superior enzyme homologues that ablated the synthesis of the side-product, and improved molar conversion of norlaudanosoline to dihydrosanguinarine to 10%.

Engineering biosynthesis of the anticancer alkaloid noscapine in yeast.

Noscapine is a potential anticancer drug isolated from the opium poppy Papaver somniferum, and genes encoding enzymes responsible for the synthesis of noscapine have been recently discovered to be clustered on the genome of P. somniferum. Here, we reconstitute the noscapine gene cluster in Saccharomyces cerevisiae to achieve the microbial production of noscapine and related pathway intermediates, complementing and extending previous in planta and in vitro investigations. Our work provides structural validation of the secoberberine intermediates and the description of the narcotoline-4'-O-methyltransferase, suggesting this activity is catalysed by a unique heterodimer. We also reconstitute a 14-step biosynthetic pathway of noscapine from the simple alkaloid norlaudanosoline by engineering a yeast strain expressing 16 heterologous plant enzymes, achieving reconstitution of a complex plant pathway in a microbial host. Other engineered yeasts produce previously inaccessible pathway intermediates and a novel derivative, thereby advancing protoberberine and noscapine related drug discovery.

Inhibition of dopamine biosynthesis by tetrahydropapaveroline.

Tetrahydropapaveroline (THP) at 5-15 microM has been found to induce L-DOPA-induced oxidative apoptosis in PC12 cells. In this study, the inhibitory effects of THP on dopamine biosynthesis in PC12 cells and tyrosine hydroxylase (TH) activity in bovine adrenal were investigated. Treatment of PC12 cells with THP at 2.5-10 microM significantly decreased the intracellular dopamine content in a concentration-dependent manner (21.3% inhibition at THP 10 microM). The activity of TH was also inhibited by the treatment with THP at 2.5-10 microM (23.4% inhibition at 10 microM). In addition, THP had an inhibitory effect on bovine adrenal TH (IC50 value, 153.9 microM). THP exhibited uncompetitive inhibition on bovine adrenal TH with a substrate l-tyrosine with the Ki value with L-tyrosine of 0.30 mM. Treatment with L-DOPA at 20-50 microM increased the intracellular dopamine content in PC12 cells and the increase in dopamine content by L-DOPA was in part inhibited when L-DOPA (20 and 50 microM) was associated with THP at non-cytotoxic (5-10 microM) or cytotoxic (15 microM) concentration ranges. However, the reduction of dopamine content by THP (15 microM) or THP (15 microM) associated with L-DOPA (20 and 50 microM) in PC12 cells was inversed by the antioxidant N-acetyl-cysteine (0.1mM). These results indicate that THP at 5-10 microM decreases the basal dopamine content and reduces the increased dopamine content induced by L-DOPA in part by the inhibition of TH activity, and that THP at 15 microM does by oxidative stress in PC12 cells.

Mammalian morphine: de novo formation of morphine in human cells.

BACKGROUND: Morphine, the major alkaloid of opium of Papaver somniferum, is one of the strongest analgesic compounds known. "Endogenous morphine" has been long isolated and authenticated by mass spectrometry in trace amounts from animal and human specific tissue or fluids. The most widely accepted explanation presently is that morphine detected in human and animal tissue is of exogenous sources (e.g. dietary origin). MATERIAL/METHODS: The biosynthetic experiments were performed with human neurobalstoma cells (SH-SY5Y) and human pancreas carcinoma (DAN-G) cells. The application experiments were done in the presence of isotopically labeled potential precursors such as (18)O(2) and [ring-(13)C(6)]-L-tyrosine. Benzylisoquinoline alkaloids present in mammalian cells were identified by GC-MS/MS. RESULTS: Growth of the SH-SY5Y cells in the presence of (18)O2 led to [(18)O(2)]-labeled morphine and [(18)O(2)]-labeled reticuline, each had a molecular mass four mass units higher than if grown in (16)O(2), indicating the presence of two atoms of (18)O per molecule. DAN-G cells cultured in an (18)O(2) atmosphere produced (S)-[ (18)O(2)]-norlaudanosoline and (S)-[ (18)O(2)]-reticuline, each labeled with (18)O atoms at only two of the four possible positions. Feeding of [ring-(13)C(6)]-tyramine, (S)-[1-(13)C, N-(13)CH(3)]-reticuline and [N-CD(3)]-thebaine to SH-SY5Y cells led each to the labeling of morphine, as established by GC-MS/MS. CONCLUSIONS: Taken together, morphine, reticuline and norlaudanosoline are unequivocally biosynthesized by cultured human cells. The precursors of morphine in the human cell lines were conclusively shown to be oxygen, tyramine, reticuline and thebaine.

Endogenous formation of morphine in human cells.

Morphine is a plant (opium poppy)-derived alkaloid and one of the strongest known analgesic compounds. Studies from several laboratories have suggested that animal and human tissue or fluids contain trace amounts of morphine. Its origin in mammals has been believed to be of dietary origin. Here, we address the question of whether morphine is of endogenous origin or derived from exogenous sources. Benzylisoquinoline alkaloids present in human neuroblastoma cells (SH-SY5Y) and human pancreas carcinoma cells (DAN-G) were identified by GC/tandem MS (MS/MS) as norlaudanosoline (DAN-G), reticuline (DAN-G and SH-SY5Y), and morphine (10 nM, SH-SY5Y). The stereochemistry of reticuline was determined to be 1-(S). Growth of the SH-SY5Y cell line in the presence of (18)O(2) led to the [(18)O]-labeled morphine that had the molecular weight 4 mass units higher than if grown in (16)O(2), indicating the presence of two atoms of (18)O per molecule of morphine. Growth of DAN-G cells in an (18)O(2) atmosphere yielded norlaudanosoline and (S)-reticuline, both labeled at only two of the four oxygen atoms. This result clearly demonstrates that all three alkaloids are of biosynthetic origin and suggests that norlaudanosoline and (S)-reticuline are endogenous precursors of morphine. Feeding of [ring-(13)C(6)]-tyramine, [1-(13)C, N-(13)CH(3)]-(S)-reticuline and [N-CD(3)]-thebaine to the neuroblastoma cells led each to the position-specific labeling of morphine, as established by GC/MS/MS. Without doubt, human cells can produce the alkaloid morphine. The studies presented here serve as a platform for the exploration of the function of "endogenous morphine" in the neurosciences and immunosciences.

Dopaminergic neurotoxins, 6,7-dihydroxy-1-(3', 4'-dihydroxybenzyl)-isoquinolines, cause different types of cell death in SH-SY5Y cells: apoptosis was induced by oxidized papaverolines and necrosis by reduced tetrahydropapaverolines.

Dopamine-derived 6,7-dihydroxy-1-(3', 4'-dihydroxybenzyl)-isoquinolines, papaverolines and tetrahydropapaverolines, have been proposed to be neurotoxin candidates related to the pathogenesis of Parkinson's disease. In this paper, the cytotoxicity of papaverolines and their N-methyl derivatives was examined using human dopaminergic neuroblastoma SH-SY5Y cells as a model of dopamine neurons. Apoptotic and necrotic cell death were assessed by morphological observation of cells after staining with propidium iodide and Hoechst 33342. Papaveroline and N-methyl-papaveroline induced apoptosis in almost all the cells with typical features of condensed and fragmented nuclei. On the other hand, (R)- and (S)-tetrahydropapaveroline caused necrosis in cells. Tetrahydropapaverolines markedly reduced adenosine triphosphate (ATP) level, whereas papaverolines did not, suggesting that the types of cell death induced by these isoquinolines, necrosis and apoptosis, depend on ATP concentrations in the cells.

Inhibition of catechol-O-methyltransferase by 6,7-dihydroxy-3,4-dihydroisoquinolines related to dopamine: demonstration using liquid chromatography and a novel substrate for O-methylation.

We report that 6,7-dihydroxy-3,4-dihydroisoquinolines related to dopamine are potent inhibitors of catechol-O-methyltransferase (COMT), but are not apparent substrates for the enzyme in vitro or in vivo. Three dihydroxy (catecholic) dihydroisoquinolines, including the 1-benzyl (DesDHP) and the 1-methyl (DSAL) analogs, were found to inhibit COMT activity in rat liver supernatant more effectively than the well-known inhibitor, tropolone. Inhibition of O-methylation was uncompetitive with substrate, and O-methylated products of the catecholic dihydroisoquinolines were undetectable. For these in vitro studies, a facile liquid chromatographic assay was developed utilizing as a site-specific substrate, 1-methyl-6,7-dihydroxy-tetrahydroisoquinoline-1-carboxylate (salsolinol-1-carboxylate). This catechol produces only one phenolic product isomer when incubated with liver supernatant and S-adenosylmethionine. Following central injection of DSAL in rats, inhibition of brain COMT in vivo was indicated by the reduced brain levels of homovanillic acid, but not of 3,4-dihydroxyphenylacetic acid. Furthermore, O-methylated DSAL metabolites could not be detected in brain by liquid or gas chromatography. We suggest that 6,7-dihydroxy-dihydroisoquinolines are "nonmethylatable" COMT inhibitors because they exist as quinoidal tautomers resembling pyridones or tropolones rather than as catechols. Quinoid formation is supported by the fluorescence and ultraviolet spectra for DSAL and its O-methyl derivatives. The experiments reveal a new class of COMT inhibitors that may be of pharmacological and mechanistic value. Additionally, 3,4-dihydroisoquinolines could arise endogenously via oxidation of the 1,2,3,4-tetrahydroisoquinolines which are ingested or produced from cellular catecholamine condensations. However, it is unlikely that dihydroisoquinoline (e.g., DSAL) concentrations necessary to inhibit COMT significantly would be attained via endogenous pathways.