NSRRC Activity Report 2022

052 NSRRC ACTIVITY REPORT 2022 presence of yatein. The resultant electron density map of the DPS•Fe•succinate•(±)-yatein complex is of sufficient quality to enable the three moieties of yatein—namely the benzodioxole ring (the A- and B-rings), lactone ring (the D-ring), and phenyl ring (the E-ring)—to be placed unambiguously in the enzyme’s substrate binding site. Because a racemic mixture of yatein was used in the crystallization experiments, (−)-yatein and (+)-yatein were individually tested to construct a ligand model for the substrate binding site to assess which isomer was present in the crystals. The ligand fitting results revealed that both enantiomers can be docked into the electron density map, which suggests that (−)-yatein and (+)-yatein may coexist in the crystals ( Figs. 2(a) and 2(b) ). Furthermore, the analysis of the binding mode of each enantiomer revealed that both enantiomers exhibit a U-shaped conformation, which not only places C7 ' at a suitable distance for hydrogen atom transfer to activate the target C–H bond but also brings C6 close enough to C7 ' for subsequent cyclization ( Figs. 2(c) and 2(d) ). This finding explains why DPS can act on both substrate enantiomers despite the difference in their stereochemistry. Concerning the catalytic mechanism of DPS, the research team performed enzyme activity assays by employing multiple substrate analogs to explore their effects on the cyclization of yatein. In contrast to the originally proposed pathway, which involves a hydroxylated intermediate, 4 the experimental results indicated that an on-pathway benzylic carbocation was likely to trigger the C–C bond formation between C6 and C7 ' . After the cyclization of yatein to deoxypodophyllotoxin, a new chiral center is formed at C7 ' . Notably, this newly formed chiral center is always in the same stereochemical configuration, regardless of which substrate enantiomer is used in the reaction. To address the question of whether the formation of different cyclized products was possible, molecular docking was performed for analysis. Assuming that their D-rings would occupy the same site as that of yatein, four possible cyclized products were manually docked into the active site ( Figs. 2(e)– 2(h) ). The results revealed that DPS imposed stereochemical constraints on the configuration of the product. Only the product whose C7 ' atom is in the R configuration could be accommodated in the active site and therefore produced ( Figs. 2(e) and 2(f) ). Thus, because of the steric constraints imposed by DPS, cyclized products have the same configuration at the C7 ' position. Fig. 2 : (a,b) Unbiased mF o − DF c electron density map of the substrate contoured at 3σ (green mesh) fitted with (−)-yatein and (+)-yatein. (c,d) Distances are shown between C6–C7 ' and C7 ' –iron in DPS-bound (−)-yatein and (+)-yatein. (e–h) Docking of four possible cyclized products into the active site. (−)-Deoxypodophyllotoxin ( 7a ) and (−)-isodeoxypodophyllotoxin ( 12a ) are produced after the ring closure of (−)-yatein. In the case of (+)-yatein, (+)-isodeoxypodophyllotoxin ( 8a ) and (+)-deoxypodophyllotoxin ( 11a ) are possible cyclized products. The black dashed lines represent distances between atoms. The coordination bonds formed by iron are represented as gray lines. [Reproduced from Ref. 5] In summary, the unprecedented substrate-bound DPS structure not only provides a structural explanation for the substrate promiscuity of DPS but also demonstrates the effect of the steric constraints imposed by DPS to control the stereochemistry of cyclized products. The results of this study provides a basis for the future application of DPS to synthesize PPT derivatives. (Reported by Min-Hao Wu, National Taiwan University) This report features the work of Nei-Li Chan and his collaborators published in PNAS 119 , e2113770119 (2022). TPS 05A Protein Microcrystallography TLS 15A1 Biopharmaceuticals Protein Crystallography • XPS, Protein Crystallography • Biological Macromolecules, Protein Structures, Life Science (a) (c) (e) (g) (b) (d) (f) (h)