title: The structural basis of an NADP⁺-independent dithiol oxidase in FK228 biosynthesis.
abstract: 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.
authors.affiliation: 1] State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China [2].
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: 1] Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA [2].
authors.forename: Cheng
authors.initials: C
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Zhi-Min
authors.initials: ZM
authors.lastname: Zhang
authors.affiliation: 1] Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA [2] UNT System College of Pharmacy, University of North Texas Health Science Center, Forth Worth, TX 76107, USA.
authors.forename: Yi-Qiang
authors.initials: YQ
authors.lastname: Cheng
authors.affiliation: 1] State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China [2] Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, 185 East Lake Road, Wuhan 430071, China.
authors.forename: Jiahai
authors.initials: J
authors.lastname: Zhou
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D055162:Biocatalysis; D002861:Chromobacterium / Q000201:enzymology* / Q000378:metabolism; D018360:Crystallography, X-Ray; D047630:Depsipeptides / Q000096:biosynthesis* / Q000737:chemistry; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000378:metabolism
chemical_list: D047630:Depsipeptides; D011994:Recombinant Proteins; D009249:NADP; C087123:romidepsin; D050862:Oxidoreductases Acting on Sulfur Group Donors; D013880:Thioredoxin-Disulfide Reductase