Functional characterization of tlmH in Streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insight into tallysomycin biosynthesis and affording a novel bleomycin analog

Abstract

Tallysomycins (TLMs) belong to the bleomycin (BLM) family of anticancer antibiotics and differ from the BLMs principally by the presence of a 4-amino-4,6-dideoxy-L-talose attached to C-41 of the TLM backbone as part of a glycosylcarbinolamide. To facilitate an understanding of the differences in anticancer activities observed between TLMs and BLMs, we thought to generate des-talose TLM analogs by engineering TLM biosynthesis in Streptoalloteichus hindustanus E465-94 ATCC 31158. Here we report (i) the engineering of the DeltatlmH mutant SB8005 strain that produces the two TLM analogs, TLM H-1 and TLM H-2, (ii) production, isolation, and structural elucidation of TLM H-1 and TLM H-2 by NMR and mass spectroscopic analyses as the desired des-talose TLM analogs, and (iii) comparison of the DNA cleavage activities of TLM H-1 with selected TLMs and BLMs. These findings support the previous functional assignment of tlmH to encode an alpha-ketoglutarate-dependent hydroxylase and unveil the TlmH-catalyzed hydroxylation at both C-41 and C-42 and the TlmK-catalyzed glycosylation of a labile carbinolamide intermediate as the final two steps for TLM biosynthesis. TlmH is apparently distinct from other enzymes known to catalyze carbinolamide formation. The availability of TLM H-1 now sets the stage to study the TlmH enzymology in vitro and to elucidate the exact contribution of the l-talose to the anticancer activities of TLMs in vivo.

Fig. 1

Structures of selected members of the bleomycin family of antitumor antibiotics BLM A2, BLM B2, TLM A, TLM B, and TLM S10b.

Fig. 2

Inactivation of tlmH by in-frame deletion in S. hindustanus. (A) Construction of the ΔtlmH mutant SB8005 strain and restriction map of the S. hindustanus wild-type and SB8005 strains showing fragment sizes upon PshAI digestion. (B) Southern analysis of the ΔtlmH mutant (lanes 3–8, six SB8005 isolates) and S. hindustanus wild-type (lane 2) genomic DNA digested with PshAI using a 1.6-kb PCR-amplified fragment as a probe. Lane 1, molecular weight marker. (C) HPLC analysis of TLM production in the S. hindustanus wild-type and the ΔtlmH mutant SB8005 strains. Symbols: ●, TLM A; ○, TLM B; ♦, TLM H-1; ♢, TLM H-2.

Fig. 3

The structures of (A) TLM H-1 supported by key ¹H - ¹H COSY and HMBC correlations and (B) TLM H-2 deduced on the basis of LC-ESI-MS analysis.

Fig. 4

DNA cleavage activities of TLM H-1 in comparison with TLM A, BLM A2, and BLM B2 as observed in plasmid relaxation and linearization assays with pBluescript II SK(+). Form A, supercoiled plasmid DNA; form B, open-circular plasmid DNA; form C, linearized plasmid DNA.

Fig. 5

The revised pathway for TLM biosynthesis featuring TlmH-catalyzed hydroxylation of both C41 and C42 to afford labile carbinolamide intermediates and TlmK-catalyzed subsequent glycosylation of their hemiaminal hydroxyl groups as the final two steps.