Hydroxyl Regioisomerization of Anthracycline Catalyzed by a Four-Enzyme Cascade

Hydroxyl regioisomerization of anthracycline catalyzed by a four-enzyme cascade

Zhuan Zhanga,1, Yu-Kang Gonga,1, Qiang Zhoua,YuHua, Hong-Min Maa, Yong-Sheng Chena, Yasuhiro Igarashib, Lifeng Pana,2, and Gong-Li Tanga,2

aState Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China; and bBiotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan

Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved January 4, 2017 (received for review June 21, 2016) Ranking among the most effective anticancer drugs, anthracyclines Kosinostatin (KST, 1), a rare C-4 deoxyanthracycline antibiotic represent an important family of aromatic polyketides generated by produced by marine Micromonospora (M.) sp. TP-A0468, exhibits type II polyketide synthases (PKSs). After formation of polyketide strong cytotoxicity against various cancer cell lines and an inhibition cores, the post-PKS tailoring modifications endow the scaffold with toward Gram-positive bacteria (11). During our previous studies, various structural diversities and biological activities. Here we demon- the anthracycline intermediate 4, bearing a C-1 hydroxyl group in strate an unprecedented four-enzyme-participated hydroxyl regioiso- D-ring, was identified from the kstB1-inactivation mutant strain merization process involved in the biosynthesis of kosinostatin. First, (Fig. 1B) (12). The high sequence identity between KST gene KstA15 and KstA16 function together to catalyze a cryptic hydroxyl- cluster and other anthracycline gene clusters indicates that KST may ation of the 4-hydroxyl-anthraquinone core, yielding a 1,4-dihydroxyl generate the C-4 hydroxyl anthracycline as primary anthracycline product, which undergoes a chemically challenging asymmetric core; in addition, our labeled acetate feeding experiments further reduction-dearomatization subsequently acted by KstA11; then, KstA10 confirmed the prediction that KST has undergone similar assembly catalyzes a region-specific reduction concomitant with dehydration to process to afford C-4 hydroxyl anthracycline (Fig. 1B) (12). If this is afford the 1-hydroxyl anthraquinone. Remarkably, the shunt product the case, an apparent hydroxyl regioisomerization of anthracycline identifications of both hydroxylation and reduction-dehydration reac- must be involved in the KST biosynthesis, which is a chemically tions, the crystal structure of KstA11 with bound substrate and challenging transformation process. Here, we report a four-enzyme- cofactor, and isotope incorporation experiments reveal mechanistic insights into the redox dearomatization and rearomatization steps. catalyzed regioselective hydroxylation-dehydroxylation process result- These findings provide a distinguished tailoring paradigm for type II ing in the hydroxyl regioisomerization, which is totally distinct from PKS engineering. currently known tailoring steps in type II PKS platforms. Results biosynthesis | C-4 deoxyanthracycline | two-component hydroxylase | NmrA-like short-chain dehydrogenase | dehydroxylation Cryptic Hydroxylation by KstA15/KstA16. Bioinformatic analysis of KST gene cluster revealed two closely linked genes, kstA15 and kstA16. The gene product of former is evolutionarily related to nthracycline antibiotics, including doxorubicin, daunorubi- polyketide cyclase, and the latter encodes a short-chain alcohol cin, idarubicin, epirubicin, and aclacinomycin (Fig. 1A), A dehydrogenase. Furthermore, the KstA15/KstA16 pair is homol- rank among the most effective anticancer drugs. However, large- ogous to the AclR/AclQ and SnoaL2/SnoaW couples, which are scale clinical application of them is often hampered by the risk of inducing cardiomyopathy (1). Hence, there have been intensive Significance attempts to develop analogs with an improved therapeutic index. In the last 50 y, most modifications have been focused on the sugar moiety, providing a successful drug, epirubicin, as well as several Enzymatic modifications of anthracycline antibiotics are urgently promising candidates under clinical trials (1, 2). In contrast, only a needed in the fields of biosynthesis, biocatalysis, and even medi- cal chemistry. However, neither hydroxyl regioisomerization nor few analogs have been generated by altering the anthracycline dehydroxylation of anthracycline core was described previously. core, most of which were obtained by chemical synthesis. There- Here, we discover an unprecedented hydroxyl regioisomerization fore, more efforts toward analogs, especially through enzymatic process in the biosynthesis of a rare carbon-4 deoxyanthracycline, modifications of the previously seldom-touched anthracycline core, which includes three tailoring steps performed by a four-enzyme are still urgently needed. cascade: two-component hydroxylases mediated a cryptic hydrox- Naturally occurring anthracycline antibiotics, usually produced ylation, and two NmrA-like short-chain dehydrogenase/reductases by Streptomyces, belong to a family of aromatic polyketides bio- catalyzed a reduction-dearomatization followed by a reduction- synthesized by type II polyketide synthases (PKSs) (3). During the dehydration process. This study expands the enzymology and last 3 decades, biosynthetic studies of this system in bacteria have chemistryoftypeIIpolyketidesynthase and provides tools to gained a deeper insight into the anthracycline’s molecular logic generate more analogs by engineering or enzymatic semisynthesis. and enzymatic mechanism (4–6). In general, the nascent poly- β-ketone generated by minimal PKS undergoes carbon-9 (C-9) Author contributions: G.-L.T. designed research; Z.Z., Y.-K.G., Q.Z., Y.H., H.-M.M., and Y.-S.C. reduction first and then cyclizations and dehydrations mediated by performed research; Y.I. contributed new reagents/analytic tools; Z.Z., Y.-K.G., L.P., and G.-L.T. analyzed data; and Z.Z., L.P., and G.-L.T. wrote the paper. cyclases, followed by oxidation of the second ring to afford the The authors declare no conflict of interest. anthraquinone portion presented in all anthracyclines. The final hydrolytic release of the anthraquinone produces the nascent This article is a PNAS Direct Submission. Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, anthracycline core, which contains a C-4 hydroxyl group in D-ring www.pdb.org (PDB ID code 5F5L and 5F5N). (Fig. 1A) (3, 7). The anthracycline core is then decorated with 1Z.Z. and Y.-K.G. contributed equally to this work. various tailoring enzymes to generate types of anthracycline anti- 2 – To whom correspondence may be addressed. Email: [email protected] or [email protected]. biotics (8 10). Therefore, most of the anthracyclines possess a C-4 cn. hydroxyl group, and a very few C-4 deoxyanthracyclines have been This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. isolated in nature. 1073/pnas.1610097114/-/DCSupplemental.

1554–1559 | PNAS | February 14, 2017 | vol. 114 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1610097114 Downloaded by guest on September 30, 2021 (Fig. 3A). The newly formed compound 5, displaying an obvious A O CO2CH3 O OH O red-shift on the UV/Vis spectrogram (Fig. 2B) and increased mo- 1 12 11 10 R 9 2 OH lecular weight by 16 Da compared with substrate 3,isexactlyin D C B A OH O OH O agreement with the expected 1,4-dihydroxy-anthracycline product 5 7 OH O OH O 4 6 (Fig. 2C). Structural elucidation of 5 provided by preparative en- R O OH O OH 1 OH O zymatic reactions confirmed 5 to be the 1,4-dihydroxy-anthracycline O N O O OH O O product (SI Appendix,NoteS6). This evidence suggests KstA15/ H N 2 O O KstA16 function together to catalyze the C-1 hydroxylation. OH HO To further explore the hydroxylation mechanism, KstA15 and Doxorubicin, R =OCH ,R =OH; H N HO 1 3 2 2 O KstA16 were purified under anaerobic conditions and performed Daunorubicin, R1 =OCH3,R2 =H; O Idarubicin, R1 =H,R2 =H. Epirubicin Aclacinomycin A the same enzymatic reaction anaerobically. Surprisingly, no pro- O duction of 5 was observed, but a new compound 3d with a de- B HN creased mass of 16 Da appeared, which mainly depended on the 10 x MCoA NH N PKS-II O OH 4 7 N O OH O O O D O 10 A 9 1 OH O O O Isoquinocycline B O OH O O (1a) O O S ACP 2 OH Kosinostatin (KST, 1)

OH O OH OH O OHOH 4 4 7 7 D A ??? D A 10 9 OH 10 OH 1 1 O OH O O OCH O OCH 3 3 4 3

Fig. 1. Representative anticancer antibiotics of anthracycline family. (A) Struc- tures of doxorubicin-like drugs and aclacinomycin A. (B) Biosynthetic pathway of kosinostatin (KST).

also encoded by adjacent genes in the aclacinomycin and noga- B lamycin biosynthetic gene clusters, respectively (SI Appendix,Fig. S1). AclR or SnoaL2 was previously discovered to be involved in the production of 1,4-dihydroxylated anthracyclines in vivo; however, in vitro experiment using AclR or SnoaL2 or cell-free lysate showed no bioconversion result (13). In addition, 1,4-dihydroxy-anthracyclines were also isolated from the aclacinomycin- producing strain (14). This evidence strongly implies that KstA15

and KstA16 are likely both involved in affording 1,4-dihydroxy- BIOCHEMISTRY anthracycline, a possible intermediate relating to the bioconversion from 3 to 4 (Fig. 1B). To evaluate their functions, kstA15 and kstA16 were inactivated individually by gene replacement (SI Ap- C pendix,Fig.S2). The resultant mutant strains M. TG1711 (ΔkstA15) and M. TG1712 (ΔkstA16) exhibited similar production profiles: both abolished the production of KST and the isomer isoquinocycline (1a) while accumulating two compounds, 3 and 3g (Fig. 2 AIII and AIV). Evaluations of the NMR and MS spectra (SI Appendix, Note S1 and Note S2) led to the successful identification of their chemical structures, both of which bear a C-4 hydroxyl group in the D-ring while being distinct from each other in the C-7 modification, where 3g is glycosylated with a γ-branched octose (Fig. 2C). The isolation of 3 and 3g, combining with characterized 4 and 4g (Fig. 2 AII and C, SI Appendix,NoteS4), leads to a con- firmation that 3 is biosynthesized by the PKS as the first anthracycline core, and subsequently is modified into 4 as second anthracycline scaffold by unknown tailoring steps (Fig. 1B). Im- portantly, KstA15 and KstA16 may perform the first step of modification and synergistically catalyze 1-hydroxylation of 3 to yield 1, 4-dihydroxy-anthracycline. Fig. 2. Characterization of the biosynthetic intermediates in vivo. (A)HPLC analysis (UV at 254 nm) of KST relative metabolites by Micromonospora sp. To validate this hypothesis, KstA15 and KstA16 were expressed strains. (I) Wild-type TP-A0468. (II)MutantTG1708(ΔkstB1). (III)Mutant and purified as His6-tagged proteins (SI Appendix,Fig.S4)andthen TG1711 (ΔkstA15). (IV)MutantTG1712(ΔkstA16). (V)MutantTG1713 incubated with 3 and NADH (or NADPH). Subsequent HPLC (ΔkstA11). (VI) Mutant TG1714 (ΔkstA10). UV-Vis analysis (B) and chemical analysis showed a new peak appears dependent on both enzymes structures (C) of anthracycline metabolites produced by the mutants.

Zhang et al. PNAS | February 14, 2017 | vol. 114 | no. 7 | 1555 Downloaded by guest on September 30, 2021 A B Reduction-Dearomatization by KstA11. Our in vitro results un- equivocally demonstrated that KstA15/A16 synergistically catalyze hydroxylation of 3 to produce 5, which may serve as an intermediate during the hydroxyl regioisomerization process; thus, a dehydroxylation must be involved in the transformation from 5 to 4. To the best of our knowledge, two enzymatic dehydroxylation pathways of aromatic ring had been reported: Birch-like one electron reduction mechanism, as exemplified by 4-hydroxybenzoyl-CoA reductase, which is a Mo-flavo-Fe/S-dependent protein (16, 17), and reduction-dehydration process-mediated dehydroxylation in the fungal melanin biosynthetic pathway, operated by 1,3,6,8-tetradroxynaphthalene reductase, 1,3,8-tridroxy- C naphthalene reductase, both belonging to the short-chain dehydrogenase/reductase (SDR) superfamily, and scytalone dehydratase (18–20). However, bioinformatic analysis revealed that neither Mo-flavo-Fe/S protein nor scytalone dehydratase homolog has been found in the KST pathway. Although we identified several genes encoding SDR family proteins within the kst cluster, sequential analysis cannot provide any clue about enzymes catalyzing dehydroxylation owing to the reaction flexibility of the SDR family (21). Thus, many efforts have been made to search for gene inactivation mutants producing the same metabolite 5. Fortunately, 5 and its glycosylated relative 5g were both observed from mutant M.TG1713(ΔkstA11) (Fig. 2 AV and C, SI Appendix,Fig.S3), implying that KstA11 may perform the next step of tailoring modification, using 5 as the substrate. Sequence analysis shows that KstA11 belongs to the NADB_ Rossmann superfamily, bearing a conserved NAD(P)H binding motif (GxxGxxG), it also displays a high similarity with the NmrA family transcriptional regulator (22). To characterize the enzymatic function, substrate 5 was incubated with KstA11 purified from Escherichia coli (SI Appendix,Fig.S8) in the presence of NAD(P)H. Interestingly, different products were detected with prolonged reaction time (Fig. 4A). The intermediate 6a,witha Fig. 3. In vitro biochemical characterization of KstA15/A16-catalyzed hy- mass increase by 2 Da compared with substrate 5, exhibits dif- droxylation. HPLC analysis (UV at 254 nm) of normal enzymatic reaction ferent UV/Vis absorption characteristics, and Vismax of 6a falls (A) and anaerobic reaction (B). (C) Proposed enzymatic mechanism. between 5 and 6b, indicating a middle conjugated degree (Fig. 4E). The final product, 6b, showing a totally different UV/Vis spectral property (Fig. 2B) and the same molecular weight as 6a,is presence of KstA16 and NAD(P)H (Fig. 3BII–V). Further struc- coincident with the dihydro-anthracycline. Structural elucidation of tural elucidation by the NMR and MS showed that 3d was a C-7 metastable 6b provided by preparative enzymatic reactions con- dehydroxy-anthracycline shunt product (Fig. 3C, SI Appendix, firms 6b to be the 5,12-dihydroxy-2,3-tetrahydro-1,4-dione 3g Note S3). In addition, when was incubated with KstA15/KstA16 product (Fig. 2C, SI Appendix, Note S8). Unfortunately, other 3d and NAD(P)H anaerobically, the same enzymatic product was enzymatic intermediates are too labile and quickly change into observed through deglycosylation instead of dehydroxylation (Fig. 6b during the extraction process, thereby preventing further 3BI). We also tested the formation of H2O2 to identify the existence purification and structural characterization. Collectively, our of peroxyanthracycline intermediate. Expectedly, H2O2 can be well enzymatic results preliminarily demonstrated that KstA11 cata- observed either in the KstA16 or the KstA16/KstA15-coupled re- lyzes the dearomatization of 5 to afford 6b, indicating the action system; moreover, the KstA16 reaction system generated dehydroxylation in KST biosynthesis is operated by a reductase- more H2O2 than the latter (SI Appendix,Figs.S5andS6). dehydratase pair. When we further investigated the enzymatic mechanism, a similar two-component monooxygenase SnoaW/SnoaL2 involved Reduction-Dehydration by KstA10. Previous studies showed that 6b in nogalamycin biosynthesis was reported, and a reaction model was unstable and could convert into 5 at room temperature was proposed (15). Our experiment results under anaerobic con- spontaneously. After optimizing the handing procedure, we de- ditions, together with their proposal, led to replenishing the hy- tected the accumulation of 6b as the major metabolite from mutant droxylation mechanism; the process resembles the reaction ΔkstA10 (Fig. 2AVI, SI Appendix,Fig.S3). To verify the function of catalyzed by flavoenzymes, where the reduced flavin cofactor ac- KstA10, we performed the biochemical assay of purified tivates dioxygen to yield a semiquinone-superoxide radical pair recombinant protein (SI Appendix,Fig.S10), using 6b as substrate (Fig. 3C). KstA16 first uses NAD(P)H to reduce the quinone (Fig. 5A). Unexpectedly, the final product 4, together with a shunt moiety of the anthracycline; the reduced substrate then reacts with product 4d, could be formed only in the presence of NAD(P)H dioxygen to form highly reactive hydroperoxide intermediate, (Fig. 5BI). Structural elucidation confirmed 4d withthesamehy- which then undergoes protonation in the presence of KstA15; the droxyl modification of aromatic ring as 4, but dehydroxylation at resulting hydroperoxide intermediate undergoes elimination of C-7 (Fig. 5A, SI Appendix, Note S5). These results preliminarily water to give a ketone followed by a tautomerization to afford the revealed that KstA10 catalyzes a reduction reaction followed by a final 1,4-dihydroxylated product. Anaerobically, the resulted anion dehydration process to rearomatize the D-ring, although less than delocalized into C-7 through resonance over the aromatic ring 10% of 4 was produced after an overnight incubation, and more system, eliminating the hydroxyl of 3 or the glycosyl group of 3g to than 70% of 6b was converted into 5 spontaneously (Fig. 5BII and give 3d (Fig. 3C). BIII). Given that 6a is more likely to be adopted by KstA10 than

1556 | www.pnas.org/cgi/doi/10.1073/pnas.1610097114 Zhang et al. Downloaded by guest on September 30, 2021 final product 4 within 1 h (Fig. 5C). Thus, the function of KstA10 as the third tailoring step was fully established in this hydroxyl regioisomerization reaction. KstA10 is another NmrA-like enzyme belonging to the SDR family with a conserved NAD(P)H binding motif (GxxGxxG) (21, 22). Further analysis indicates that KstA10 is homologous to + NAD -dependent nucleotide diphosphate sugar epimerases (23), which act on totally different substrates from that in KST biosynthesis. In addition, scytalone dehydratase in the fungal melanin

B C

D BIOCHEMISTRY

Fig. 4. Biochemical and structural characterizations of KstA11-catalyzed reduction-dearomatization. (A) HPLC analysis (UV at 254 nm) of enzymatic reaction with NADH. (B) The combined ribbon representation and stick model showing F the overall structure of KstA11 in complex with cofactor NAD+ and substrate. The bound cofactor NAD+ and substrate are shown in the stick model. (C)The combined surface charge potential representation (contoured at ±4 kT/eV; blue/ + red) and the stick model showing the detailed interactions of KstA11 with NAD and substrate. (D) Stereo view showing the detailed interactions among key + residues of KstA11, NAD , and substrate. The hydrogen bonds involved in the binding are shown as dotted gray lines. (E) UV/Vis analysis of the reaction. (F) Proposed chemical reaction. (G) Proposed reaction mechanism from 5a to 6a.

Fig. 5. Biochemical assay of KstA10-catalyzed dehydration-rearomatization. (A) Chemical reactions. HPLC analysis (UV at 254 nm) of enzymatic reactions 6b, then we performed consecutive reactions in which the product using 6b as the substrate by KstA10 (B)and5 as the substrate by KstA11/ of KstA11 acts as the substrate for KstA10 (Fig. 5A). Just as KstA10 (C). (D) Proposed reaction mechanism. Comparison of the 1H-NMR expected, more than 90% of substrate 5 was converted into the (I)and13C-NMR (II)spectraof2H-6b with 6b (E)and2H-4 with 4 (F).

Zhang et al. PNAS | February 14, 2017 | vol. 114 | no. 7 | 1557 Downloaded by guest on September 30, 2021 biosynthesis is cofactor independent (18, 19). However, evidence Appendix,Fig.S7B). Subsequently, mutations that are expected to obtained from our biochemical assays suggested that the de- weaken the interaction between KstA11 and NADH, such as R37E hydration catalyzed by KstA10 is dependent on NAD(P)H and N154K, or interaction between KstA11 and substrate 5,suchas (SI Appendix,Fig.S11). To gain further insights into the enzymatic I252Y, all dramatically decrease the catalytic activity (SI Appendix, + mechanism, KstA11 and KstA10 assays were conducted in the Figs. S8 and S9). In addition, the conformations of 5 and NAD in presence of (S)-[4-2H]NADPH obtained by glucose dehydrogenase the complex are further stabilized by a π-π stacking between 5 and + from Bacillus megaterium DSM 2894 BmGDH, using D-[1-2H] the nicotinamide group of NAD , as well as a hydrogen bond be- + glucose as the deuterium donor (24, 25), as the proS hydrogen is tweentheC-4oxygenof5 and the 2′-OH group of NAD (Fig. 4D). the hydrogen transferred to the substrate during reduction ob- Given the unique chemical structure of 5,thetransitionstatefor served from the cocrystal structure of KstA11 in complex with 5 6b + the KstA11 catalyzed dearomatization reduction of to would substrate 5 and NAD (see Structural Insight into the KstA11-Cat- be expected to proceed from the 1,4-diketo tautomer of 5 (here- alyzed Reaction). Expectedly, a 1-Da shifted fragment ion at m/z after referred to as 5a)(Fig.4F). In the complex, 5a is highly 416.1103 was detected in a KstA11/BmGDH coupled assay (SI preferred, as the oxygen atoms located at the C-1 and C-4 posi- Appendix,NoteS9), indicating a deuterium incorporation in 6b; tions of 5 are well positioned to form two hydrogen bonds with the 2 + large-scale reactions were then performed to isolate H-6b and side chain hydroxyl group of Y264 and the 2′-OH of NAD ,re- + elucidate the exact deuterium incorporation position. A compari- spectively. Meanwhile, the 2′-OH of NAD also forms a hydrogen 1 13 2 son of H-NMR and C-NMR spectra of H-6b and 6b showing bond with K129, which is further hydrogen bonded with the E90 2 that C-2 of H-6b has been split into three peaks (a triplet) and the residue to couple with a water molecule (Fig. 4D). Therefore, relative hydrogen peak area has decreased confirmed the deuterium 2 there is a hydrogen bonding network that proceeds from the water incorporation at C-2 of H-6b (Fig. 5 D and E, SI Appendix,Note to the C-4 oxygen of substrate 5. On the basis of the aforemen- S9). A 1-Da shifted fragment ion at m/z 398.0997 and a 2-Da shifted tioned biochemical and structural data, we proposed an enzymatic fragment ion at m/z 399.1065werebothobservedintheKstA11/ mechanism for the KstA11-catalyzed reduction of 5 to 6b (Fig. 4 F KstA10/BmGDH coupled assay, implying that more than 1 deute- and G). When substrate 5 and cofactor NADH are in complex rium had been incorporated into 4 (SI Appendix,NoteS10). Simi- with KstA11, the specific interactions among them not only induce larly, we prepared 2H-4 by large-scale enzymatic reaction and 2 1 13 the close contact between 5 and NADH but also stabilize the 1,4- elucidated H-4 by H-NMR and C-NMR. A head-to-head diketo 5a.Then,theproS hydrogen of NADH (the hydrogen that comparison of 1H-NMR and 13C-NMR spectra of 2H-4 and 4 2 is transferred as a hydride to the substrate in the reduction re- showed the peak disappearance of C-4 and relative H-4 of H-4 and action) is directed toward the C-2 position of 5a, as the C-4 of the + a peak attenuation of C-2 and relative H-2 (1/2 peak area), dem- nicotinamide ring of NAD is highly close to the C-2 carbon of onstrating the absolute deuterium incorporation at C-4 and partial 2 5 in the complex structure (Fig. 4D). Importantly, aforemention- deuterium incorporation at C-2 in H-4 (Fig. 5 D and F, SI Ap- ed deuteration-based labeling studies confirmed this type of pendix,NoteS10). According to the identification of shunt product 1,4-addition reaction (Fig. 5D). Meanwhile, the 2′-OH of NADH, and deuterium isotope experiments, we proposed that KstA10 which is fed by the hydrogen bond network that includes K129, functions as a ketoreductase and dehydratase, catalyzing a 1,2-nu- E90, and a water molecule, serve as the ultimate proton donor to cleophilic addition, followed by dehydration to aromatize the D-ring. the C-4 oxygen of 5a to generate the product 6a (Fig. 4G). Finally, First, KstA10 binds 6a as the real substrate and subsequently 6a is converted to 6b through a chemical rearrangement because isomerizes it into 6b in the active site; next is the nucleophilic ad- of the lower energy level of conjugated system in the latter (Fig. dition of proS hydrogen of NADPH to keto-carbonyl of C-4 to form 4F). In line with this proposal, the substitution of the key catalytic a tetrahedral intermediate. Then the intermediate undergoes elim- K129 residue with Arg essentially abolished the enzymatic activity ination of 4-OH, concomitant with hydrogen transferring from of KstA11 (SI Appendix,Fig.S9). Interestingly, the E90A muta- 12-OH to carbonyl of C1. Finally, deprotonation of 5-OH accom- tion has little effect on the enzyme activity, suggesting the K129 panied with leaving hydride at C-2 gives the final product 4.In contrast, the formation of 4d may result from deprotonation of may directly couple with a water molecule in the proton relay in 5-OH concomitant with leaving 7-OH, and then deprotonation at this mutant. C-2 accompanied with protonation of C-7 (Fig. 5D). Discussion Structural Insight into the KstA11-Catalyzed Reaction. To gain mecha- Combining in vivo genetic characterizationandinvitrobiochemical nistic insight into the unusual reaction catalyzed by KstA11, we studies, we uncovered an unprecedented hydroxyl regioisomeriza- determined the crystal structure of KstA11 in complex with substrate tion process operated on the aromatic ring of anthracycline leading + 5 and a relevant cofactor NAD to 1.3-Å resolution (SI Appendix, to the 1-hydroxyl anthraquinone, an essential intermediate pro- Table S1). In the complex structure, two KstA11 molecules pack posed to be a prerequisite for the spirocyclic formation during KST together to form a symmetric dimer, and each monomer contains 12α-h elixes (α1−α12) and 9 β-strands (β1−β9) and binds with one + substrate 5 and one NAD molecule in a 1:1:1 stoichiometry (Fig. OH O OHOH OH O OHOH O OH + KstA15/A16 4 4B). Notably, 5 and NAD directly contact with each other and pack 4 4 KstA11

NAD(P)H 1 1 extensively with a solvent-exposed elongated groove located at the 10 NAD(P) OH OH 1 middle region of KstA11 (Fig. 4 B and C). The specific interactions OH O O OH O O OCH + O OCH3 3 3 + 5 5a of KstA11 with substrate 5 and NAD are mainly mediated by hy- NAD(P)H drophobic contacts and polar interactions (Fig. 4C, SI Appendix,Fig. KstA11 S7). Specifically, the hydrophobic moiety of 5 packs extensively with NAD(P)+ the hydrophobic pocket formed by theV114, F126, F150, F155, KstA10 O OHOH H O O OH OHOH OH OH 2 or W158, L171, I246, I252, M255, F256, and F259 residues of KstA11, 4 KstA10 4 spontaneous and the oxygen located at the C-1 position of substrate 5 forms a 4 NAD(P) 1 10 NAD(P)H 1 hydrogen bond with the side chain of Y264 (SI Appendix,Fig.S7A). OH OH 1 + OH O O OH O OH O OCH3 O OCH3 In parallel, the NAD molecule forms a number of hydrogen bonds + 4 6b 6a with the T14, T16, Q17, D58, T80, K129, and N154 residues, and its aromatic adenine and nicotinamide groups form a cation-π in- Fig. 6. Summary of the hydroxyl regioisomerization performed by KstA15/ teraction with R37 and a π-π stacking with F151, respectively (SI A16-KstA11-KstA10.

1558 | www.pnas.org/cgi/doi/10.1073/pnas.1610097114 Zhang et al. Downloaded by guest on September 30, 2021 biosynthesis. This chemically challenging transformation has gone aflatoxin B1 biosynthesis expanded the substrates to hydroxynaph- through a hydroxylation and a selective dehydroxylation by two thoquinones and tricyclic anthrahydroquinones (30–33). The dis- tandem dearomatization and rearomatization processes, which in- covery of KstA11-catalyzed regio- and stereoselective hydrogenation clude three tailoring steps acting on the anthracycline core: the of tetracyclic anthracyclines and regioselective dehydroxylation by primary core structure 3 synthesized by the PKS is catalyzed by KstA11/KstA10 couple are remarkable, which indicates that addi- KstA15/KstA16 to generate a 1, 4-dihydroxyl intermediate 5;KstA11 tional enzymes with broad substrate spectrum are waiting to be performs an asymmetric dearomatization, using 5 as substrate to explored for biocatalysts. Significantly, as a tailoring modification yield 6a;and6a can isomerize into 6b intheactivesiteofKstA10, acting on the anthracycline core, our results definitely broaden the which then undergoes regioselectively reduction-dehydration to af- tailoring spectrum to modify the previously seldom-touched ford secondary core structure 4 mediated by KstA10 (Fig. 6). anthracycline core to generate more analogs for future cancer Significantly, the dehydroxylation operated by KstA11 and drug studies. KstA10, to the best of our knowledge, is the first enzymatic system realizing the dehydroxylation on the aromatic ring of anthracycline. Materials and Methods 4-Demethoxydaunorbicin (idarubicin), a synthetic daunorubi- Materials and methods are summarized in SI Appendix, Materials and Meth- cin analog, displays superior fat solubility and cellular uptake ods. Supporting data are provided in SI Appendix, Figs. S1–S11. X-ray crystal- compared with daunorubicin or doxorubicin, indicating the lographic data collection, primers, strains and plasmids are summarized in 4-deoxyanthrancycline core is vital for its antitumor activity (26). SI Appendix,TablesS1–S3. Physicochemical characterizations of the compounds Accordingly, it is meaningful to develop an elaborate and ver- are provided in SI Appendix,NotesS1–S10. satile system of enzyme-mediated dehydroxylation on the aromatic ring of anthracycline. Our results now established feasibly ACKNOWLEDGMENTS. We thank Professor Ren-Xiao Wang of Shanghai Insti- enzymatic dehydroxylation on the aromatic ring of anthracy- tute of Organic Chemistry for guiding the protein modeling; Professor Han-Jie cline. It is promising that this dehydroxylation could be used to Ying in Nanjing University of Technology for providing the plasmid pRSF- provide aimed anthracycline analogs in the future by protein BmGDH; Professor Chang-Sheng Zhang of South China Sea Institute of Ocean- ology, Chinese Academy of Sciences, for kindly giving us D-[1-2H]-glucose; engineering. In addition, dearomatizations are chemically chal- Professor Michael Müller of Institut für Pharmazeutische Wissenschaften, Albert- lenging in a sense both of chemistry and of biology (16, 17, 27), Ludwigs-Universität Freiburg for helpful discussion, suggestion, and sharing as the asymmetric hydrogenation of aromatic compounds, which unpublished data with us; and Shanghai Synchrotron Radiation Facility beam- was considered a versatile and practical synthetic strategy line BL17U and National Center for Protein Science Shanghai Beamline 19U1 method to obtain chiral compound, has been an active and for X-ray beam time. This work was supported in part by grants from Ministry of Science and Technology (2013CB836900), National Natural Science Founda- attractive field of methodology research (28, 29). Recently, tion of China (81373307 and 31330003), Science and Technology Commission characterization of the NADPH-dependent reductases involved in of Shanghai Municipality (14XD1404500 and 15JC1400400), Chinese Academy fungal 1,8-dihydroxynaphthalene melanin, monodictyphenone, and of Sciences (XDB20020200), and the “Thousand Talents Program” (L.P.).

1. Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L (2004) Anthracyclines: Molecular 19. Thompson JE, et al. (2000) The second naphthol reductase of fungal melanin bio- advances and pharmacologic developments in antitumor activity and cardiotoxicity. synthesis in Magnaporthe grisea: Tetrahydroxynaphthalene reductase. J Biol Chem Pharmacol Rev 56(2):185–229. 275(45):34867–34872. 2. Zhang G, et al. (2005) Syntheses and biological activities of disaccharide daunorubi- 20. Liao D, Basarab GS, Gatenby AA, Valent B, Jordan DB (2001) Structures of trihy- cins. J Med Chem 48(16):5269–5278. droxynaphthalene reductase-fungicide complexes: Implications for structure-based 3. Metsä-Ketelä M, Niemi J, Mäntsälä P, Schneider G (2008) Anthracycline biosynthesis: design and catalysis. Structure 9(1):19–27. Genes, enzymes, and mechanisms. Top Curr Chem 282:101–140. 21. Kavanagh KL, Jörnvall H, Persson B, Oppermann U (2008) Medium- and short-chain 4. Shen B (2000) Biosynthesis of aromatic polyketides. Top Curr Chem 209:1–51. dehydrogenase/reductase gene and protein families : The SDR superfamily: Func- 5. Hertweck C, Luzhetskyy A, Rebets Y, Bechthold A (2007) Type II polyketide synthases: tional and structural diversity within a family of metabolic and regulatory enzymes. Gaining a deeper insight into enzymatic teamwork. Nat Prod Rep 24(1):162–190. Cell Mol Life Sci 65(24):3895–3906. 6. Das A, Khosla C (2009) Biosynthesis of aromatic polyketides in bacteria. Acc Chem Res 22. Stammers DK, et al. (2001) The structure of the negative transcriptional regulator 42(5):631–639. NmrA reveals a structural superfamily which includes the short-chain dehydrogenase/ – 7. Zhou H, Li Y, Tang Y (2010) Cyclization of aromatic polyketides from bacteria and reductases. EMBO J 20(23):6619 6626. BIOCHEMISTRY fungi. Nat Prod Rep 27(6):839–868. 23. Thoden JB, Holden HM (1998) Dramatic differences in the binding of UDP-galactose 8. Niemi J, Metsä-Ketelä M, Schneider G, Mäntsälä P (2008) Biosynthetic anthracycline and UDP-glucose to UDP-galactose 4-epimerase from Escherichia coli. Biochemistry – variants. Top Curr Chem 282:75–99. 37(33):11469 11477. 9. Rix U, Fischer C, Remsing LL, Rohr J (2002) Modification of post-PKS tailoring steps 24. Zhang G, et al. (2014) Mechanistic insights into polycycle formation by reductive cy- – through combinatorial biosynthesis. Nat Prod Rep 19(5):542–580. clization in ikarugamycin biosynthesis. Angew Chem Int Ed Engl 53(19):4840 4844. 25. Ye Q, et al. (2010) Construction and co-expression of a polycistronic plasmid encoding 10. Olano C, Méndez C, Salas JA (2010) Post-PKS tailoring steps in natural product-producing carbonyl reductase and glucose dehydrogenase for production of ethyl (S)-4-chloro-3- actinomycetes from the perspective of combinatorial biosynthesis. Nat Prod Rep 27(4): hydroxybutanoate. Bioresour Technol 101(17):6761–6767. 571–616. 26. Adams N, et al. (1990) Synthesis and antitumor activity of novel 4-demethoxyan- 11. Furumai T, Igarashi Y, Higuchi H, Saito N, Oki T (2002) Kosinostatin, a quinocycline thracyclines. J Med Chem 33(9):2375–2379. antibiotic with antitumor activity from Micromonospora sp. TP-A0468. J Antibiot 27. Eberlein C, et al. (2013) Identification and characterization of 2-naphthoyl-coenzyme (Tokyo) 55(2):128–133. A reductase, the prototype of a novel class of dearomatizing reductases. Mol 12. Ma HM, et al. (2013) Unconventional origin and hybrid system for construction of Microbiol 88(5):1032–1039. pyrrolopyrrole moiety in kosinostatin biosynthesis. Chem Biol 20(6):796–805. 28. Zhuo CX, Zhang W, You SL (2012) Catalytic asymmetric dearomatization reactions. 13. Beinker P, et al. (2006) Crystal structures of SnoaL2 and AclR: Two putative hydrox- Angew Chem Int Ed Engl 51(51):12662–12686. ylases in the biosynthesis of aromatic polyketide antibiotics. J Mol Biol 359(3): 29. Wang DS, Chen QA, Lu SM, Zhou YG (2012) Asymmetric hydrogenation of hetero- – 728 740. arenes and arenes. Chem Rev 112(4):2557–2590. 14. Oki T, et al. (1979) Antitumor anthracycline antibiotics, aclacinomycin a and ana- 30. Schätzle MA, et al. (2012) Tetrahydroxynaphthalene reductase: Catalytic properties of – logues. II. Structural determination. J Antibiot (Tokyo) 32(8):801 819. an enzyme involved in reductive asymmetric naphthol dearomatization. Angew 15. Siitonen V, Blauenburg B, Kallio P, Mäntsälä P, Metsä-Ketelä M (2012) Discovery of a Chem Int Ed Engl 51(11):2643–2646. two-component monooxygenase SnoaW/SnoaL2 involved in nogalamycin bio- 31. Husain SM, Schätzle MA, Lüdeke S, Müller M (2014) Unprecedented role of hydro- synthesis. Chem Biol 19(5):638–646. naphthoquinone tautomers in biosynthesis. Angew Chem Int Ed Engl 53(37): 16. Unciuleac M, Warkentin E, Page CC, Boll M, Ermler U (2004) Structure of a xanthine 9806–9811. oxidase-related 4-hydroxybenzoyl-CoA reductase with an additional [4Fe-4S] cluster 32. Schätzle MA, Husain SM, Ferlaino S, Müller M (2012) Tautomers of anthrahy- and an inverted electron flow. Structure 12(12):2249–2256. droquinones: Enzymatic reduction and implications for chrysophanol, mono- 17. Boll M (2005) Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like dictyphenone, and related xanthone biosyntheses. JAmChemSoc134(36): reductions. Biochim Biophys Acta 1707(1):34–50. 14742–14745. 18. Basarab GS, et al. (1999) Catalytic mechanism of scytalone dehydratase: Site-directed 33. Conradt D, Schätzle MA, Haas J, Townsend CA, Müller M (2015) New insights into the mutagenisis, kinetic isotope effects, and alternate substrates. Biochemistry 38(19): conversion of versicolorin A in the biosynthesis of aflatoxin B1. J Am Chem Soc 6012–6024. 137(34):10867–10869.

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