Tandem Enzymatic Oxygenations in Biosynthesis of Epoxyquinone Pharmacophore of Manumycin-type Metabolites Zhe Rui, Moriah Sandy, Brian Jung, Wenjun Zhang

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Tandem Enzymatic Oxygenations in Biosynthesis of Epoxyquinone Pharmacophore of Manumycin-type Metabolites Zhe Rui, Moriah Sandy, Brian Jung, Wenjun Zhang Chemistry & Biology Volume 20, Issue 7, Pages 879-887 (July 2013) DOI: 10.1016/j.chembiol.2013.05.006 Copyright © 2013 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2013 20, 879-887DOI: (10. 1016/j. chembiol. 2013 Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 Chemical Structures of Manumycin-Type Compounds Asukamycin is A1; protoasukamycin is C1; 4-hydroxyprotoasukamcyin is D1. See also Figure S1. Chemistry & Biology 2013 20, 879-887DOI: (10.1016/j.chembiol.2013.05.006) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Characterization of AsuA2, AsuC11, and AsuC12 (A) AsuA2 activity toward various aromatic substrates in ATP-[32P]PPi exchange assays. A relative activity of 100% for 3,4-AHBA-dependent exchange corresponds to 82k CPM. All chemical structures are shown in Figure S3. (B) Determination of AsuA2 kinetic parameters by ATP-[32P]PPi exchange assays. Error bars represent SDs from at least three independently performed experiments. (C) Detection of holo-AsuC12 without AsuA2 and 3,4-AHB-S-AsuC12 in the AsuA2 reaction by HRMS with deconvolution. (D) Schematic of 3,4-AHBA activation and loading. See also Figures S3 and S4. Chemistry & Biology 2013 20, 879-887DOI: (10.1016/j.chembiol.2013.05.006) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 Characterization of AsuE1 (A) Extracted ion chromatograms showing the conversion of C1 to D1 catalyzed by AsuE1. The calculated mass with 10 ppm mass error tolerance was used. (B) Determination of AsuE1 kinetic parameters. Error bars represent SDs from at least three independently performed experiments. See also Figures S5 and S6. Chemistry & Biology 2013 20, 879-887DOI: (10.1016/j.chembiol.2013.05.006) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 Characterization of AsuE2 (A) Determination of AsuE2 kinetic parameters. Error bars represent SDs from at least three independently performed experiments. (B) Extracted ion chromatograms showing the conversion of C1 to D1 catalyzed by AsuE1/E2. The calculated mass with 10 ppm mass error tolerance was used. Assays contain 100 μM NADH, 100 μM protoasukamycin, 1 μM AsuE1, and 1 μM AsuE2. The control group lacks AsuE2 but contains 0.5 nM exogenous FMN. See also Figure S7. Chemistry & Biology 2013 20, 879-887DOI: (10.1016/j.chembiol.2013.05.006) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 5 Characterization of AsuE3 (A) Extracted ion chromatograms showing the conversion of D1 to A1 catalyzed by AsuE3. The calculated mass with 10 ppm mass error tolerance was used. (B) Determination of AsuE3 kinetic parameters. Error bars represent SDs from at least three independently performed experiments. See also Figures S6 and S8. Chemistry & Biology 2013 20, 879-887DOI: (10.1016/j.chembiol.2013.05.006) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 6 Schematic of the Epoxyquinone Moiety Formation in Asukamycin See also Figure S2 and Table S1. Chemistry & Biology 2013 20, 879-887DOI: (10.1016/j.chembiol.2013.05.006) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 7 HPLC Analysis of Asukamycin Derivatives in ΔasuA3 Supplemented with 3-ABA, 4-ABA, 4-HBA, and 3,4-ACBA R′ = -H in C12 and C15; R′ = -COCH3 in C11 and C16; R = R1–R5 in C6–C10; λ = 325 nm. ΔasuA3, 3,4-AHBA deficient mutant. See also Figures S9–S11. Chemistry & Biology 2013 20, 879-887DOI: (10.1016/j.chembiol.2013.05.006) Copyright © 2013 Elsevier Ltd Terms and Conditions