Identification of (2S,3S)-β-Methyltryptophan as the Real Biosynthetic Intermediate of Antitumor Agent Streptonigrin

Abstract

Streptonigrin is a potent antitumor antibiotic, active against a wide range of mammalian tumor cells. It was reported that its biosynthesis relies on (2S,3R)-β-methyltryptophan as an intermediate. In this study, the biosynthesis of (2S,3R)-β-methyltryptophan and its isomer (2S,3S)-β-methyltryptophan by enzymes from the streptonigrin biosynthetic pathway is demonstrated. StnR is a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase that catalyzes a transamination between L-tryptophan and β-methyl indolepyruvate. StnQ1 is an S-adenosylmethionine (SAM)-dependent C-methyltransferase and catalyzes β-methylation of indolepyruvate to generate (R)-β-methyl indolepyruvate. Although StnR exhibited a significant preference for (S)-β-methyl indolepyruvate over the (R)-epimer, StnQ1 and StnR together catalyze (2S,3R)-β-methyltryptophan formation from L-tryptophan. StnK3 is a cupin superfamily protein responsible for conversion of (R)-β-methyl indolepyruvate to its (S)-epimer and enables (2S,3S)-β-methyltryptophan biosynthesis from L-tryptophan when combined with StnQ1 and StnR. Most importantly, (2S,3S)-β-methyltryptophan was established as the biosynthetic intermediate of the streptonigrin pathway by feeding experiments with a knockout mutant, contradicting the previous proposal that stated (2S,3R)-β-methyltryptophan as the intermediate. These data set the stage for the complete elucidation of the streptonigrin biosynthetic pathway, which would unlock the potential of creating new streptonigrin analogues by genetic manipulation of the biosynthetic machinery.

Figure 1. Chemical structures of streptonigrin (STN, 1), lavendamycin (2), streptonigron (3), and 3′-desmethylstreptonigrin (4).

Figure 2. HPLC profiles of biochemical assays of StnR, StnQ1, and StnK3.

(a) (i) l-Trp; (ii) β-methyl indolepyruvate; (iii) indolepyruvate; (iv) StnR incubated with l-Trp and α-KG; (v) StnQ1 incubated with indolepyruvate and SAM. (b) (i) l-Trp; (ii) (2S,3S)-β-MeTrp; (iii) (2S,3R)-β-MeTrp; (iv) StnR/Q1 incubated with l-Trp and SAM; (v) StnR/K3/Q1 incubated with l-Trp and SAM. (c) (i) (2S,3R)-β-MeTrp; (ii) (2S,3S)-β-MeTrp; (iii) StnR (20 μM) incubated with racemic β-methyl indolepyruvate and l-Trp; (iv-viii) StnR (2 μM) incubated with racemic β-methyl indolepyruvate and l-Trp for 0.5 h (iv), 1.0 h (v), 2.0 h (vi), 4.0 h (vii), 8.0 h (viii); (ix) l-Trp. ♦ S-adenosylhomocysteine (SAH) confirmed by MS.

Figure 3. HPLC profiles of fermentation extracts.

(i) STN-producing strain wild-type; (ii) ΔstnQ1 mutant; (iii) ΔstnQ1 mutant fed with (2S,3R)-β-MeTrp; (iv) ΔstnQ1 mutant fed with (2S,3S)-β-MeTrp. ♦ 1 and ◊ 4; • is not STN confirmed by MS analysis.

Figure 4. StnR/K3/Q1-catalyzed biosynthesis of (2S,3R)- and (2S,3S)-β-MeTrp from L-Trp and the modified biosynthetic pathway of STN.