The structural basis of an NADP⁺-independent dithiol oxidase in FK228 biosynthesis

The disulfide bond is unusual in natural products and critical for thermal stability, cell permeability and bioactivity. DepH from Chromobacterium violaceum No. 968 is an FAD-dependent enzyme responsible for catalyzing the disulfide bond formation of FK228, an anticancer prodrug approved for the treatment of cutaneous T-cell lymphoma. Here we report the crystal structures of DepH and DepH complexed with a substrate analogue S,S'-dimethyl FK228 at 1.82 Å and 2.00 Å, respectively. Structural and biochemical analyses revealed that DepH, in contrast to the well characterized low molecular weight thioredoxin reductases (LMW TrxRs), is an NADP⁺-independent dithiol oxidase. DepH not only lacks a conserved GGGDXAXE motif necessary for NADP⁺ binding in the canonical LMW TrxRs, but also contains a 11-residue sequence which physically impedes the binding of NADP⁺. These observations explain the difference between NADP⁺-independent small molecule dithiol oxidases and NADP⁺-dependent thioredoxin reductases and provide insights for understanding the catalytic mechanism of dithiol oxidases involved in natural product biosynthesis.