NSRRC Activity Report 2022

Life Science 051 Structural Basis Underlying the Catalytic Mechanism of Deoxypodophyllotoxin Synthase Deoxypodophyllotoxin synthase (DPS) catalyzes a crucial cyclization reaction to produce the fused tetracyclic structure of podophyllotoxin. This study demonstrates the catalytic characteristics of DPS from a structural perspective. P odophyllotoxin (PPT) is a naturally occurring aryltetralin lignan and is primarily obtained through extraction from the dried roots and rhizomes of Podophyllum species, such as Sinopodophyllum hexandrum and Podophyllum peltatum . 1 PPT is of pharmaceutical interest because it can interfere with the association between α-tubulin and β-tubulin to disrupt microtubule assembly, 2 which leads to cell cycle arrest in the G2/M phase. Therefore, PPT is regarded as a promising compound for inhibiting abnormal cell proliferation. Furthermore, its glycoside derivatives, including etoposide and teniposide ( Fig. 1(a) ), exhibit potent anticancer activity through a distinct mode of action. Specifically, these drugs are capable of insertion into the type II topoisomerase (Top2)-mediated DNA cleavage site to interrupt the catalytic cycle of Top2, 3 which results in the accumulation of DNA double-strand breaks and subsequent apoptotic cell death. Given their outstanding antiproliferative effect, PPT derivatives have been used extensively in cancer therapy. Because PPT is a valuable starting material for the semisynthesis of anticancer drugs, its demand has increased over time. Thus, meeting the commercial need for PPT by merely obtaining PPT from natural resources is now difficult. Consequently, alternative methods—including plant cultivation, in vitro cell culture, total synthesis, and chemoenzymatic synthesis—have been developed to increase PPT production. Among these methods, chemoenzymatic synthesis, which is achieved using enzymes involved in the biosynthesis of PPT to overcome critical steps in compound synthesis, has emerged as an attractive approach. Deoxypodophyllotoxin synthase (DPS) catalyzes a chemically challenging stereospecific cyclization reaction to convert yatein into deoxypodophyllotoxin in the biosynthetic pathway of PPT ( Fig. 1(b) ). On the basis of sequence similarity and the cofactors required by DPS to catalyze the reaction, DPS is classified as a member of the iron- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase superfamily. 4 Although DPS has been utilized in the synthesis of deoxypodophyllotoxin analogs, how DPS recognizes its substrates and performs a stereospecific cyclization reaction remains unclear, and one of the major impediments to understanding the DPS-catalyzed mechanism is the lack of structural information about DPS. For the Fe/2OG oxygenase superfamily, no substrate-bound structural information regarding the enzymes that catalyze ring formation, especially through C–C bond formation, has been obtained. Fig. 1 : (a) Structures of PPT ( 1 ), etoposide ( 2 ), teniposide ( 3 ), and etopophos ( 4 ). (b) DPS catalyzes the conversion of (−)-yatein ( 6a ) into deoxypodophyllotoxin ( 7a ). Asterisk ( ✽ ) labels represent the chiral centers. [Reproduced from Ref. 5] (a) (b) To investigate the detailed catalytic mechanism of DPS-catalyzed cyclization, Nei-Li Chan (National Taiwan University) and his collaborators Tun-Cheng Chien (National Taiwan Normal University) and Wei-chen Chang (North Carolina State University, USA) determined the crystal structures of DPS in multiple stages of its catalytic cycle. 5 Specifically, X-ray diffraction data were collected at TLS 15A1 and TPS 05A of the NSRRC. The DPS•Fe•2OG and DPS•Fe•succinate•(±)-yatein structures were characterized at resolutions of 2.09 and 2.05 Å, respectively. Similar to other Fe/2OG oxygenases, DPS exhibits a conserved core structure featuring a double-stranded β-helix fold and an active site harboring a 2-His-1-carboxylate facial triad for iron coordination. In addition, studies have revealed that DPS exhibits certain substrate promiscuity. 6 Specifically, DPS can catalyze its native substrate, namely (−)-yatein, and substrate enantiomer, namely (+)-yatein, regardless of the opposite stereochemistry at C8 and C8 ' . To elucidate the structural basis of this phenomenon, Chan’s team crystallized DPS in the