Structural characterization of DynU16, a START/Bet v1-like protein involved in dynemicin biosynthesis

Alvarado, Sarah; Miller, Mitchell D.; Bhardwaj, Minakshi; Thorson, Jon S.; Lanen, Steven G. Van; Phillips, G.N.

doi:10.1107/s2053230x21008943

Cited by 1 publication

(2 citation statements)

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“…Overlaying these protein structures with the Orf14 predicted structure revealed the prominent conservation of the core fold that binds a structurally diverse class of hydrophobic ligands ranging from polyketide, polyene, and lipids to small aromatic rings, steroids, and hormones; for example, type II PKS ARO/CYL BexL (PDB:4XRW) involved in the regiospecific cyclization of the precursor of the anticancer agent BE-7585A, abscisic acid receptor PYL (PDB:7MLD), a pathogenesis-related class 10 protein LIPR10.2B (PDB:5MXB) that binds melatonin, thebaine synthase 2 (PDB:6KA3) that converts (7S)-salutaridinol 7- O -acetate to thebaine, the key component in codeine and morphine biosynthesis, the isomerase LFS (PDB:5GTG) involved in the natural production of the lachrymatory agent syn -propanethial S-oxide, and plant allergen Pru av 1 (PDB:1E09). However, more interesting is the structural homology to two proteins, the proposed self-sacrifice resistance protein CalU16 (PDB:4FPW) from calicheamicin 2 biosynthesis and the biosynthetic protein DynU16 (PDB:6V04) from the Dyn 3 BGC . CalU16 is a small monodomain protein containing the conserved core SRBPCC fold, that is proposed to bind the enediyne skeleton of 2 .…”

Section: Resultsmentioning

confidence: 99%

“…32 However, more interesting is the structural homology to two proteins, the proposed self-sacrifice resistance protein CalU16 (PDB:4FPW) from calicheamicin 2 biosynthesis 33 and the biosynthetic protein DynU16 (PDB:6V04) from the Dyn 3 BGC. 34 CalU16 is a small monodomain protein containing the conserved core SRBPCC fold, that is proposed to bind the enediyne skeleton of 2. DynU16 is a hetero-didomain protein, strikingly similar to the organized portion of the Orf14 model (Figure 6b).…”

Section: ■ Introductionmentioning

confidence: 99%

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Examining Heterodimerization by Aryl C–N Coupling in Dynemicin Biosynthesis

Alley

Townsend

2023

ACS Chem. Biol.

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Distinct among the enediyne antitumor antibiotics, the dynemicin subgroup is comprised of two discrete halves, an enediyne and an anthraquinone, but each is ultimately derived from the same linear β-hydroxyhexaene precursor. The linkage of these two halves by an aryl C–N bond is examined here using a variety of experimental approaches. We demonstrate that this heterodimerization is specific for anthracenyl iodide as the corresponding bromo- and amino-substituted anthracenes do not support dynemicin biosynthesis. Furthermore, biochemical experiments and chemical model reactions support an SRN1 mechanism for the aryl C–N coupling in which electron transfer occurs to the iodoanthracene, followed by loss of an anthracenyl iodide and partition of the resulting aryl radical between C–N coupling and reduction by hydrogen abstraction. An enzyme pull-down experiment aiming to capture the protein(s) involved in the coupling reaction is described in which two proteins, Orf14 and Orf16, encoded by the dynemicin biosynthetic gene cluster, are specifically isolated. Deletion of orf14 from the genome abolished dynemicin production accompanied by a 3-fold increased accumulation of the iodoanthracene coupling partner, indicating the plausible involvement of this protein in the heterodimerization process. On the other hand, the deletion of orf16 only reduced dynemicin production to 55%, implying a noncatalytic, auxiliary role of the protein. Structural comparisons using AlphaFold imply key similarities between Orf14 and X-ray crystal structures of several proteins from enediyne BGCs believed to bind hydrophobic polyene or enediyne motifs suggest Orf14 templates aryl C–N bond formation during the central heterodimerization in dynemicin biosynthesis.

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