Functional characterization of tlmK unveiling unstable carbinolamide intermediates in the tallysomycin biosynthetic pathway

Abstract

Tallysomycins (TLMs) belong to the bleomycin family of anticancer antibiotics. TLMs differ from bleomycins primarily by the presence of a 4-amino-4,6-dideoxy-l-talose sugar attached to C-41 as part of a glycosylcarbinolamide. We previously proposed, on the basis of bioinformatics analysis of the tlm biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158, that the tlmK gene is responsible for the attachment of this sugar moiety. We now report that inactivation of tlmK in S. hindustanus abolished TLM A and TLM B production, the resultant DeltatlmK mutant instead accumulated five new metabolites, and introduction of a functional copy of tlmK to the DeltatlmK mutant restored TLM A and TLM B production. Two major metabolites, TLM K-1 and TLM K-2, together with three minor metabolites, TLM K-3, TLM K-4, and TLM K-5, were isolated from the DeltatlmK mutant, and their structures were elucidated. These findings provide experimental evidence supporting the previous functional assignment of tlmK to encode a glycosyltransferase and unveil two carbinolamide pseudoaglycones as key intermediates in the TLM biosynthetic pathway. TlmK stabilizes the carbinolamide intermediates by glycosylating their hemiaminal hydroxyl groups, thereby protecting them from hydrolysis during TLM biosynthesis. In the absence of TlmK, the carbinolamide intermediates fragment to produce an amide TLM K-1 and aldehyde intermediates, which undergo further oxidative fragmentation to afford carboxylic acids TLM K-2, TLM K-3, TLM K-4, and TLM K-5.

FIGURE 1.

Structures of selected members of the BLM family of antitumor antibiotics BLM A2, BLM B2, TLM A, and TLM B.

FIGURE 2.

Inactivation of tlmK and genetic complementation to the ΔtlmK in-frame deletion mutant. A, construction of the ΔtlmK mutant strain SB8003 and restriction maps of the S. hindustanus wild-type and SB8003 mutant strains as well as the pBS8012 cosmid carrying the ΔtlmK in-frame deletion mutation upon XcmI digestion. B, Southern analysis of the ΔtlmK mutant strain SB8003 (lanes 1, 2, 3, 4, and 5 for five independent isolates) with the ΔtlmK mutant construct pBS8012 (lane 6) and tlmK wild-type construct pBS8008 (lane 7) as controls upon XcmI digestion and using the 2.2-kilobase (kb) PCR-amplified fragment from the wild-type strain as a probe. C, HPLC analysis of (a) TLM production in S. hindustanus wild type, (b) new metabolite accumulation in the ΔtlmK mutant strain SB8003, and (c) restoration of TLM production in the ΔtlmK-complemented recombinant strain SB8004. ○, TLM A; □, TLM B; •, TLM K-1; ▪, TLM K-2; ▴, TLM K-3; ♦, TLM K-4; ▾, TLM K-5. mAU, milliabsorbance.

FIGURE 3.

Key ¹H-¹H COSY and heteronuclear multiple bond correlation (HMBC) correlations of TLM K-1, K-2, and K-5.

FIGURE 4.

Biosynthesis of TLMs featuring the carbinolamide pseudoaglycones as key intermediates, which in the absence of the TlmK glycosyltransferase, undergo rapid degradation into TLM K-1, K-2, K-3, K-4, and K-5.

FIGURE 5.

Selected natural products containing a carbinolamide moiety (boxed).

FIGURE 6.

Cleavage of the break light by TLM A, TLM K-1, and TLM K-2 assayed according to the literature procedure (39). DNA cleavage was followed over time of assays containing 3.2 nm break light and TLM A (•, 200 nm), TLM K-1 (▴, 2 μm), TLM K-2 (2 μm, data not shown), or no drug as a control (×) in 25 mm Tris-HCl (pH 7.5) at 37 °C. TLM K-2, whose data were omitted from the plot for clarity, was identical to the no drug control.