Enzymatic Synthesis of the Ribosylated Glycyl-Uridine Disaccharide Core of Peptidyl Nucleoside Antibiotics

Transcription

1 SUPPRTING INFRMATIN Enzymatic Synthesis of the Ribosylated Glycyl-Uridine Disaccharide Core of Peptidyl Nucleoside Antibiotics Zheng Cui, Xiaodong Liu, Jonathan verbay, Wenlong Cai, Xiachang Wang,, Anke Lemke, Daniel Wiegmann, Giuliana Niro, Jon S. Thorson,, Christian Ducho and Steven G. Van Lanen* Department of Pharmaceutical Sciences and Center for Pharmaceutical Research and Innovation, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States Jiangsu Key Laboratory for Functional Substance of Chinese Medicine, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing , People s Republic of China Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2 3, Saarbrücken, Germany TABLE F CNTENTS Page Table S1. Amino donor substrates for Mur20 S2 Figure S1. Sequence alignments S3, S4 Figure S2. In vitro characterization of Mur16 S5 Figure S3. Mass spectrum of 10c S6 Figure S4. 1 NMR spectrum (D 2, 400 Mz) and 13 C NMR spectrum (D 2, 100 Mz) of 10c S7 Figure S5. PLP dependency of Mur17 S8 Figure S6. In vitro characterization of Mur20 S9 Figure S7. Stimulation of Mur20 activity with PLP S10 Figure S8. Mass spectrum of 17a S11 Figure S9. 1 NMR spectrum (D 2, 100 Mz) of 17a S12 Figure S C NMR spectrum (D 2, 100 Mz) of 17a S13 Figure S11. SQC NMR spectrum (D 2, 400 Mz) of 17a S13 Figure S CSY NMR spectrum (D 2, 400 Mz) of 17a S14 Figure S13. In vitro characterization of Mur19 with an alternative sugar donor S15 Figure S14. 1 NMR spectrum (D 2, 400 Mz) of AQC-modified 17b S16 Figure S CSY NMR spectrum (D 2, 400 Mz) of AQC modified 17b S17 Figure S16. Characterization of Mur19 with potential alternative sugar acceptors S18 Figure S17. Potential reversibility of Mur19 S19 Figure S18. Mass spectrum of 18 S20 Figure S19. 1 NMR spectrum (D 2, 400 Mz) of 18 S21 Figure S C NMR spectrum (D 2, 100 Mz) of 18 S22 Figure S CSY spectrum (D 2, 400 Mz) of 18 S23 Figure S22. SQC spectrum (D 2, 400 Mz) of 18 S24 Figure S23. MBC spectrum (D 2, 400 Mz) of 18 S25 Figure S24. 1 NMR spectrum (D 2, 400 Mz) of 11a S26 Figure S25. 1 NMR spectrum (D 2, 400 Mz) of 12 S26 Figure S26. 1 NMR spectrum (D 2, 400 Mz) of 13a S27 Figure S27. 1 NMR spectrum (D 2, 400 Mz) of 13b S27 S1

2 Table S1. Amino donor substrates for Mur20. Amino Donor Relative Specific Activity (%) a Lip with L-Met 260 L-Tyr 100 L-Trp 71 L -Arg 69 L-Met 65 S-adenosyl-L-methionine 55 L-Phe 45 L-Ala 30 L-is 21 L-Gln 19 L-Lys 19 a Activity with other proteinogenic amino acids was not detected. S2

3 A B C S3

4 D E F Figure S1. Sequence alignments of the putative proteins involved in the biosynthesis of the ADR- GlyU disaccharide of muraymycin and the homologous Lip enzyme previously determined to be involved in 4 biosynthesis. (A) Alignment of Mur16 with LipL, a nonheme Fe(II), α-ketoglutarate (αkg):10a dioxygenase. (B) Alignment of Mur17 with LipK, a pyridoxal-5 -phosphate-dependent (PLP) L-Thr:11a transaldolase. (C) Alignment of Mur20 with Lip, a PLP-dependent 11a aminotransferase. (D) Alignment of Mur26 with LipP, a pyrimidine phosphorylase. (E) Alignment of Mur18 with LipM, a nucleotidylyltransferase. (F) Alignment of Mur19 with LipN, a ribosyltransferase. S4

5 A B R e la tiv e In te n s ity (% ) V e lo c ity ( M /m in ) m /z [A s c o rb a te ] (m M ) C D R e la tiv e In te n s ity (% ) m \z A ( - )1 0 a 1.0 (+ )A r g 0.8 ( + )1 0 a T im e (m in ) E F R e la tiv e In te n s ity (% ) R e la tiv e In te n s ity (% ) m \z m \z Figure S2. In vitro characterization of Mur16. (A) Mass spectrum for the ion peak eluting at time t = min for the Mur16-catalyzed reaction with 10a (expected (M + ) + and (M + 3) + ions at m/z = and 261.1, respectively, for 11a). (B) Dependence of the Mur16-catalyzed reaction on ascorbic acid. (C) Mass spectrum for the ion peak corresponding to the coproduct succinate (expected (M - ) - ion at m/z = for succinate). (D) Enzyme-coupled reaction monitoring the formation of succinate from the Mur16-catalyzed reaction in the presence (+) or absence (-) of 10a or substituting 10a with L-Arg. (E) Mass spectrum for the ion peak eluting at time t = min for the Mur16-catalyzed reaction with 10b (expected (M + ) + ion at m/z = for 11b). (F) Mass spectrum for the ion peak eluting at time t = min for the Mur16-catalyzed reaction with 10c (expected (M + ) + ion at m/z = for 11c). S5

6 Figure S3. Mass spectrum of 10c. RMS (ESI/Q-TF) m/z: [M - ] - Calcd for C1014N29P ; Found S6

7 Figure S4. 1 NMR spectrum (D2, 400 Mz) and 13 C NMR spectrum (D2, 100 Mz) of 10c. S7

8 A 4 A U 3 2 A U W a v e le n g th (n m ) W a v e le n g th (n m ) B a ( iii) A ( ii) ( i) E lu tio n T im e (m in ) Figure S5. PLP dependency of Mur17. (A) UV/VIS spectrum of purified Mur17. AU, absorbance units. (B) PLC analysis of 5-h incubations of 11a with (i) no enzyme, (ii) Mur17 with exogenous PLP, and (iii) Mur17 without PLP. S8

9 R e la tiv e In te n s ity (% ) R e la tiv e In te n s ity (% ) A B C m /z m /z 1 1 b 1 3 c 1 0 c A b ( ii) A c ( ii) ( i) ( i) E lu tio n T im e (m in ) E lu tio n T im e (m in ) Figure S6. In vitro characterization of Mur20. (A) Mass spectrum of Mur20-product 13a; RMS (ESI/Q-TF) m/z: [M + ] - Calcd for C914N ; Found (B) PLC analysis of 6 h incubations of 11b with (i) no enzyme and (ii) Mur20. The inset depicts the mass spectrum for the ion peak eluting at t = 7.8 min corresponding to 13b [expected (M + ) + ion at m/z = for C913N34]. (C) PLC analysis of 6 h incubations with 10c and Mur16 with (i) no additional enzyme and (ii) Mur20. The inset depicts the mass spectrum for the ion peak eluting at t = 7.8 min corresponding to 13c [expected (M + ) + ion at m/z = for C1015N35]. S9

10 A A U W a v e le n g th (n m ) B 1 3 a 1 1 a ( iii) A ( ii) ( i) E lu tio n T im e (m in ) Figure S7. Stimulation of Mur20 activity with PLP. (A) (A) UV-VIS spectrum of purified Mur20 (black line), following a 2 min incubation after the addition of L-Met (red line), and following a 2 min after the addition of L-Met and 11a (blue line). AU, absorbance units. (B) PLC analysis of 5-h incubations of 11a with (i) no enzyme, (ii) Mur20 with exogenous PLP, and (iii) Mur20 without PLP. S10

11 Figure S8. Mass spectrum of 17a. RMS (ESI/Q-TF) m/z: [M + ] + Calcd for C1625N ; Found S11

12 Figure S9. 1 NMR spectrum (D2, 100 Mz) of 17a. Figure S C NMR spectrum (D2, 100 Mz) of 17a. S12

13 Figure S11. SQC NMR spectrum (D2, 400 Mz) of 17a. S13

14 Figure S CSY NMR spectrum (D2, 400 Mz) of 17a. S14

15 A L ip P 1 3 b + U TP a n d M u r 1 8 / L ip M N P P 1 5 b N N 2 N C M u r1 9 2 N N N 1 7 b + U D P B C 1 7 b 1 4 U D P b 1 3 b U M P U TP ( iv ) A ( iii) ( ii) ( i) E lu tio n T im e (m in ) R e la tiv e In te n s ity N N N N N N C 2 N N m /z Figure S13. In vitro characterization of Mur19 with an alternative sugar donor. (A) Reaction catalyzed by Mur19 with in situ-generated 15b. (B) PLC analysis of 6 h incubations of 12 and 13b with (i) LipP, LipM, and LipN; (ii) LipP, Mur18, and LipN; (iii) LipP, LipM, and Mur19; and (iv) LipP, Mur18, and Mur19. LipN is the Mur19 ortholog involved in 4 biosynthesis and does not utilize 15b as a substrate. (C) Mass spectrum for the AQC-derivatized product eluting at t = 2.8 min, which is consistent with the molecular formula for AQC-17b (shown); RMS (ESI/Q- TF) m/z: [M + ] + Calcd for C3637N ; Found S15

16 Figure S14. 1 NMR spectrum (D2, 400 Mz) of AQC-modified 17b. S16

17 Figure S CSY NMR spectrum (D2, 400 Mz) of AQC modified 17b. S17

18 A C N N N N N N N N C N N M u ra y m y c in D 4 B Figure S16. Characterization of Mur19 with potential alternative sugar acceptors. (A) Structure of muraymycin D4. (B) Mass spectrum of muraymycin D4; RMS (ESI/Q-TF) m/z: [M + ] + Calcd for C3253N ; Found S18

19 A C N N N N N 2 N N N R N C N N M u r1 9 U D P M u r a y m y c in D N UDP R Muraymycin D1 R = Me Muraymycin D2 R = Muraymycin D3 R = B U D P u rid in e m u ra y m y c in D 3 m u ra y m y c in D 2 m u ra y m y c in D 1 (ii) (i) (ii) (i) (ii) (i) E lu tio n T im e (m in ) Figure S17. Potential reversibility of Mur19. (A) ypothetical reverse reaction catalyzed by Mur19. (B) PLC analysis of 12-h incubations of UDP and the indicated muraymycin congener with (i) no enzyme and (ii) Mur19. Low levels of uridine are generated in the presence of Mur19. S19

20 Figure S18. Mass spectrum of 18. RMS (ESI/Q-TF) m/z: [M + ] + Calcd for C1423N ; Found S20

21 Figure S19. 1 NMR spectrum (D2, 400 Mz) of 18. S21

22 Figure S C NMR spectrum (D2, 100 Mz) of 18. S22

23 Figure S CSY spectrum (D2, 400 Mz) of 18. S23

24 Figure S22. SQC spectrum (D2, 400 Mz) of 18. S24

25 Figure S23. MBC spectrum (D2, 400 Mz) of 18. S25

26 Figure S24. 1 NMR spectrum (D2, 400 Mz) of 11a. Figure S25. 1 NMR spectrum (D2, 400 Mz) of 12. S26

27 Figure S26. 1 NMR spectrum (D2, 400 Mz) of 13a. Figure S27. 1 NMR spectrum (D2, 400 Mz) of 13b. S27