Supplementary Data for TetX is a Flavin-Dependent Monooxygenase Conferring Resistance to Tetracycline Antibiotics by Wangrong Yang, Ian F. Moore, Kalinka P. Koteva, Donald W. Hughes, David C. Bareich and Gerard D. Wright. Table S1. Oligonucleotide primers for PCR amplification of TetX constructs. Restriction sites are underlined. Protein Primer Sequence TetX 5 -CCG GAA TTC AAG CTT TTA TTA TAC ATT TAA CAA TTG C 5 -CCG GAA TTC CAT ATG ACA ATG CGA ATA GAT ACA GAC TetX1 5 -GCG TCT AGA CAT ATG GCA AAC TTG TTA CAA CAA ACC GG 5 -GGA ATT CAA GCT TTT ATA CAT TCA TTA GCT GTT GAA AAG TAA AGC TetX2 tetx2f 5 -CCG GAA TTC CAT ATG ACA ATG CGA ATA GAT ACA GAC tetx2r 5 - CCG GAA TTC AAG CTT TTA T TA TAC ATT TAA CAA TTG C Table S2. 1 H NMR assignments of oxytetracycline in 0.1M DCl/D 2 O. Proton Chemical Shift (ppm) Coupling Constant (Hz) 4 4.30 J 4,4a = 1.4 4-N(CH 3 ) 2 2.98, 2.92 4a 2.87 5 3.88 J 5,4a = 11.4 5a 2.90 6-CH 3 1.72 7 7.14 J 7,9 = 0.9, J 7,8 = 7.8 8 7.49 J 8,9 = 8.5 9 6.88 33
Table S3. 13 C NMR assignments of oxytetracycline in 0.1M DCl/D 2 O. Carbon Chemical Shift (ppm) 1 192.14 2 95.47 2-CONH 2 171.66 3 186.01 4 65.71 4-N(CH 3 ) 2 42.36, 41.01 4a 41.01 5 64.27 5a 49.42 6 69.88 6-CH 3 22.67 6a 145.52 7 115.25 8 137.03 9 117.41 10 160.41 10a 113.63 11 193.50 11a 104.24 12 170.05 12a 72.38 Table S4. 1 H NMR Assignments of 11a-hydroxy-oxytetracycline-6,12-hemiketal (P1) in 0.1M DCl/D 2 O. Proton Chemical Shift (ppm) Coupling Constant (Hz) 4 3.97 J 4,4a = 1.8 4-N(CH 3 ) 2 3.15, 2.96 4a 3.67 5 4.16 J 5,4a = 8.9 5a 2.85 6-CH 3 1.57 7 7.12 J 7,8 = 7.8 8 7.60 J 8,9 = 8.4 9 7.04 34
Table S5. 13 C NMR Assignments of 11a-hydroxy-oxytetracycline-6,12-hemiketal (P1) in 0.1M DCl/D 2 O. Carbon Chemical Shift (ppm) 1 194.01 2 98.96 2-CONH 2 171.18 3 186.54 4 69.27 4-N(CH 3 ) 2 41.91 4a 43.37 5 66.33 5a 59.77 6 79.63 6-CH 3 17.23 6a 147.59 7 114.75 8 137.40 9 117.68 10 161.12 10a 113.19 11 199.51 11a 85.22 12 102.29 12a 74.10 35
Figure S1. The 1 H NMR spectrum of P1 in 0.1M DCl/D 2 O. 36
Figure S2. Expansion of the aromatic region of the 1 H NMR spectrum of P1. Figure S3. Expansion of the 1 H NMR spectrum of P1 in 0.1M DCl/D 2 O. 37
Figure S3. Expansion of the 1 H NMR spectrum of P1 showing the resonances of the four methine protons and the protons of the dimethylamino group. 38
Figure S4. Expansion of the 1 H NMR spectrum of P1 showing the resonance of the 6- CH 3 group. 39
Figure S5. The COSY spectrum of P1. 40
Figure S6. Expansion of the COSY spectrum of P1. The circled peaks indicate the coupling interaction between the doublet of doublets at 3.67 ppm and the doublets at 4.16 ppm and 3.97 ppm. 41
Figure S7. Expansion of the aromatic region of the COSY spectrum of P1. 42
Figure S8. The NOE difference spectrum of oxytetracycline generated by saturating the 6-CH 3 resonance at 1.72 ppm, enhancement of the resonances at 7.14 ppm, 3.876 ppm and 2.90 ppm can be seen. 43
Figure S9. The NOE difference spectrum of P1 generated by saturating the resonance at 1.45 ppm, enhancement of the resonances at 7.12 ppm, 4.16 ppm and 2.85 ppm can be seen. 44
Figure S10. The 13 C NMR spectrum of P1 0.1M DCl/D 2 O. 45
Figure S11. Expansion of the 13 C NMR spectrum of P1 0.1M DCl/D 2 O. 46
Figure S12. Expansion of the 13 C NMR spectrum of P1 0.1M DCl/D 2 O. 47
Figure S13. The HSQC spectrum of P1 0.1M DCl/D 2 O, ten proton-carbon correlations observed. 48
Figure S14. Expansion of the HSQC spectrum of P1 showing the correlations of the aromatic protons. 49
Figure S15. Expansion of the HSQC spectrum of P1 showing the correlations of the nonaromatic protons. The circled signals show the methine proton-carbon correlations. 50
Figure S16. The HMBC spectrum of P1 0.1M DCl/D 2 O. The three circled peaks indicate the 2-bond and 3-bond couplings of the 6-CH 3 protons to C6a, C6 and C5a. 51
Figure S17. Expansion of the HMBC spectrum of P1, the circles show the 2-bond and 3- bond correlations of the H5a proton. 52
Figure S18. Expansion of the HMBC spectrum of P1, the circle shows the 3-bond correlation to C11. 53
Figure S19. Expansion of the aromatic region of the HMBC spectrum of P1. 54
Figure S20. Purity of TetX2. SDS-Polyacrylamide gel (11%) of 4 consecutive fractions of TetX2 after purification. The gel was stained with coomassie blue. The mass of the high molecular weight markers (HMW) in kda is shown on the left. 97 84 HMW 1 2 3 4 66 55 45 36 55