Figure S1. Structure Identification of compound 2. (a) 1H NMR and 13C NMR of
compound 2. 1H NMR (600 MHz, D2O) δ 3.42-3.21 (m, 1H), δ 1.19 (d, J = 12.6, 1H),
δ 2.91 (ddd, J1=12.3, J2=9.8, J3=4.2,1H), δ 3.42-3.21 (m, 1H), δ 3.58 (dd, J1=7.0,
J2=2.0, 1H), δ 3.54 (t, J=9.2, 1H), δ 5.3 (d, J=3.8, 1H), δ 2.85 (m, 1H), δ 4.00 (ddd,
J1=10.3, J2=8.7, J3=7.5, 1H), δ 2.85-2.81 (m, 1H), δ 3.74 (dd, J1=10.1, J2=1.9, 1H), δ
1.9 (dq, J1=13.0, J2=3.9, 1H), δ 4.96 (d, J=3.8, 1H), δ 3.93 (dd, J1=12.8, J2=3.4, 1H),
δ 3.42-3.21 (m, 1H), δ 3.88 (dd, J1=10.8, J2=3.9, 1H), δ 2.97(d, J=10.8, 1H), δ 1.24 (d,
J=6.9, 3H), δ 2.63 (s, 3H), 1.14 (s, 3H). 13C NMR (151 MHz, D2O) δ 50.53, 34.07,
49.19, 86.5, 70.18, 74.08, 99.64, 54.17 (d, J=4.8), 75.18 (d, J=5.3), 72.29, 85.98,
46.39, 100.18, 67.33, 63.83, 70.74, 67.42, 15.78, 35.51, 21.11. (b) 1H-1H-COSY NMR
of 2. (c) 31P NMR of 2 (4.49 ppm, zero point calibration is 85% phosphoric acid
solution).
Figure S2. HPLC-ELSD analysis of GenP reactions with gentamicin X2 and G418. (i)
X2 standard; (ii) GenP-catalyzed reaction with X2; (iii) G418 standard; (iv)
GenP-catalyzed reaction with G418.
Figure S3. 1H and 13C NMR of compound 6. 1H NMR (600 MHz, D2O) δ 3.31-3.01
(m, 1H), δ 1.18 (d, J=12.2,1H), 2.15 (ddd, J1=10.3, J2=9.5, J3=4.0, 1H), δ 3.32-3.11
(m, 1H), δ 3.44 (d, J=4.2, 1H), δ 3.55 δ (dd, J1=7.0, J2=2.0, 1H), δ 3.23 (t, J=9.2, 1H),
δ 5.35 (d, J=2.2, 1H), δ 3.10-3.02 (m, 1H), δ 1.95 (d, J=12.2, 1H), δ 2.20 (ddd,
J1=10.5, J2=7.0, J3=4.0, 1H), δ 4.95-4.86 (m, 1H), δ 5.06 (d, J=4.0, 1H), δ 3.82 (dd,
J1=10.8, J2=3.4, 1H), δ 2.62-2.51 (m, 1H), δ 4.05 (dd, J1=10.8, J2=3.9, 1H), δ 3.31 (d,
J=10.8, 1H), δ 1.26 (d, J=6.9, 3H), δ 2.64 (s, 3H), δ 1.16 (s, 3H). 13C NMR (151 MHz,
D2O) δ 50.76, 35.78, 49.17, 99.54, 74.35, 86.31, 100.5, 58.16, 29.93, 99.3, 145.8,
197.4, 83.7, 72.1, 63.81, 71.08, 67.86, 25.49, 33.94, 21.23.
Figure S4. Ammonia analysis of GenB3-catalyzed reaction with 4.
Figure S5. GenP and GenB3 catalyzes dideoxygenation. HPLC-ELSD analysis of (i)
GenP and GenB3-catalyzed reaction (ii) GenP and GenB3-catalyzed reaction with
L-Glu, and (iii) GenP and GenB3-catalyzed reaction with α-Ketoglutarate.
Figure S6. Identification of PLP-binding sites in GenB3-catalyzed reactions. (a)
HPLC-ELSD analysis of reactions catalyzed by the following: (i) GenP with
gentamicin B1; (ii) GenP and GenB3 with gentamicin B1; (iii) GenP with G418; and
(iv) GenP and GenB3 with G418. (b) Schematic of the GenP and GenB3 in-vitro
reaction with gentamicin B1. (c) MS analysis of 20.
Figure S7. Dissection of the C-4',5' reduction process catalyzed by GenB4.
Figure S8. Schematic representation and confirmation by PCR amplication of inframe
deletion of genP and genB3.
Table S1. Kinetic constants of GenP catalyzing phosphorylation of different substrates
Table S2. List of primers used in this study
Table S3. List of strains and plasmids used in this study