Biosynthesis of camptothecin. In silico and in vivo tracer study from [1-13C]glucose

Abstract

Camptothecin derivatives are clinically used antitumor alkaloids that belong to monoterpenoid indole alkaloids. In this study, we investigated the biosynthetic pathway of camptothecin from [1-13C]glucose (Glc) by in silico and in vivo studies. The in silico study measured the incorporation of Glc into alkaloids using the Atomic Reconstruction of Metabolism software and predicted the labeling patterns of successive metabolites from [1-13C]Glc. The in vivo study followed incorporation of [1-13C]Glc into camptothecin with hairy roots of Ophiorrhiza pumila by 13C nuclear magnetic resonance spectroscopy. The 13C-labeling pattern of camptothecin isolated from the hairy roots clearly showed that the monoterpene-secologanin moiety was synthesized via the 2C-methyl-D-erythritol 4-phosphate pathway, not via the mevalonate pathway. This conclusion was supported by differential inhibition of camptothecin accumulation by the pathway-specific inhibitors (fosmidomycin and lovastatin). The quinoline moiety from tryptophan was also labeled as predicted by the Atomic Reconstruction of Metabolism program via the shikimate pathway. These results indicate that camptothecin is formed by the combination of the 2C-methyl-D-erythritol 4-phosphate pathway and the shikimate pathway. This study provides the innovative example for how a computer-aided comprehensive metabolic analysis will refine the experimental design to obtain more precise biological information.

Figure 1.

Formation of IPP and dimethylallyl diphosphate (DMAPP) via the MVA pathway (A) or the MEP pathway (B). Dots, Positions of ¹³C arising from [1-¹³C]Glc via the MEP pathway. Squares, Positions of ¹³C via the MVA pathway. Lovastatin inhibits HMG-CoA reductase, and fosmidomycin inhibits DXP reductoisomerase. DXP, 1-Deoxy-d-xylulose 5-phosphate; G3P, glyceraldehyde 3-phosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate; HMG-CoA, hydroxymethylglutaryl-CoA; MEP, 2-C-methyl-d-erythritol 4-phosphate.

Figure 2.

Overview of metabolite mappings. Ery4P, Erythrose 4-phosphate; Glu6P, Glc 6-phosphate; 3PG, 3-phosphoglycerate; PRA, 5-phosphoribosylanthranilate; Rib5P, Rib 5-phosphate; SHK, shikimic acid.

Figure 3.

Glycolysis of [1-¹³C]Glc. [1-¹³C]Glc gives [1,6-¹³C]Glc 6-phosphate through dihyroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) by the reversibility of glycolytic enzymes and triose phosphate isomerase. [3-¹³C]Pyruvate and [2-¹³C]acetyl-CoA are formed from [3-¹³C]glyceraldehyde 3-phosphate. Dots, Positions of ¹³C arising from [1-¹³C]Glc.

Figure 4.

Biosynthetic pathway of tryptamine from [1,6-¹³C]Glc 6-phosphate via the shikimate pathway. Dots, Positions of ¹³C arising from [1-¹³C]Glc. ANTH, Anthranilate; CHA, chorismic acid; CPDP, 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate; Ery4P, erythrose 4-phosphate; G3P, glyceraldehyde 3-phosphate; INGP, indole-3-glycerol phosphate; PEP, phosphoenolpyruvate; PRA, 5-phosphoribosylanthranilate; SHK, shikimic acid; Rib-5P-1PP, 5-phospho-Rib-1-pyrophosphate.

Figure 5.

Biosynthesis of camptothecin from [1-¹³C]Glc. Red dots, Positions of ¹³C arising from [1-¹³C]Glc via the MEP pathway. Pink dots, Positions of carbons randomized. Blue squares, Positions of ¹³C via the MVA pathway. Green asterisks, Position of ¹³C via the shikimate pathway. In box, possible labeling pattern of camptothecin from different pathways.

Figure 6.

125 MHz ¹³C-NMR spectra of camptothecin in dimethyl sulfoxide (DMSO). Numbers indicate the assignments of carbon positions of camptothecin. Asterisks, ¹³C-enriched signals. A, Standard camptothecin. B, ¹³C-enriched camptothecin be feeding with [1-¹³C]Glc.

Figure 7.

Effect of pathway inhibitors on camptothecin accumulation. The inhibitors, lovastatin and fosmidomycin, were added to the 3-week-old hairy roots at the final concentrations indicated. Three days after the addition of the compounds, the hairy roots were extracted for determination of camptothecin. Bar = sd of triplicate determinations. The difference of camptothecin production among control and treated samples were statistically significant by Student's t test (^**, P < 0.01).