Pyridoxal-5'-Phosphate-Dependent Enzyme GenB3 Catalyzes C-3',4'-Dideoxygenation in Gentamicin Biosynthesis
Background: C-3',4'-dideoxygenation structure in gentamicin can prevent deactivation by aminoglycoside 3'-phosphotransferase (APH(3')) in drug-resistant pathogens. However, the enzyme catalyzing the dideoxygenation step in the gentamicin biosynthesis pathway remains unknown.
Results: Here, we report GenP catalyzes 3′ phosphorylation of gentamicin biosynthesis intermediates JI-20A, JI-20Ba, and JI-20B. We further demonstrate that a pyridoxal-5′-phosphate (PLP)-dependent enzyme GenB3 uses these phosphorylated substrates to form 3',4'-dideoxy-4',5'-ene-6'-oxo products. The following C-6' transamination and GenB4 catalyzed reduction of 4',5' olefin lead to the formation of gentamicin C. To the best of our knowledge, GenB3 is the first PLP dependent enzyme catalyzing dideoxygenation in aminoglycoside biosynthesis.
Conclusions: This discovery solves the long-standing puzzle in gentamicin biosynthesis, also enriches the chemistry of PLP dependent enzymes. Interestingly, these results demonstrate that to evade APH(3') deactivation from the pathogens, the gentamicin producers evolved a smart strategy, which utilized their own APH(3') to activate hydroxyls as leaving groups for the 3',4'-dideoxygenation in gentamicin biosynthesis.
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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
Posted 22 Dec, 2020
On 10 Jan, 2021
Received 09 Jan, 2021
On 04 Jan, 2021
On 25 Dec, 2020
Invitations sent on 20 Dec, 2020
On 17 Dec, 2020
On 17 Dec, 2020
On 17 Dec, 2020
On 16 Dec, 2020
Pyridoxal-5'-Phosphate-Dependent Enzyme GenB3 Catalyzes C-3',4'-Dideoxygenation in Gentamicin Biosynthesis
Posted 22 Dec, 2020
On 10 Jan, 2021
Received 09 Jan, 2021
On 04 Jan, 2021
On 25 Dec, 2020
Invitations sent on 20 Dec, 2020
On 17 Dec, 2020
On 17 Dec, 2020
On 17 Dec, 2020
On 16 Dec, 2020
Background: C-3',4'-dideoxygenation structure in gentamicin can prevent deactivation by aminoglycoside 3'-phosphotransferase (APH(3')) in drug-resistant pathogens. However, the enzyme catalyzing the dideoxygenation step in the gentamicin biosynthesis pathway remains unknown.
Results: Here, we report GenP catalyzes 3′ phosphorylation of gentamicin biosynthesis intermediates JI-20A, JI-20Ba, and JI-20B. We further demonstrate that a pyridoxal-5′-phosphate (PLP)-dependent enzyme GenB3 uses these phosphorylated substrates to form 3',4'-dideoxy-4',5'-ene-6'-oxo products. The following C-6' transamination and GenB4 catalyzed reduction of 4',5' olefin lead to the formation of gentamicin C. To the best of our knowledge, GenB3 is the first PLP dependent enzyme catalyzing dideoxygenation in aminoglycoside biosynthesis.
Conclusions: This discovery solves the long-standing puzzle in gentamicin biosynthesis, also enriches the chemistry of PLP dependent enzymes. Interestingly, these results demonstrate that to evade APH(3') deactivation from the pathogens, the gentamicin producers evolved a smart strategy, which utilized their own APH(3') to activate hydroxyls as leaving groups for the 3',4'-dideoxygenation in gentamicin biosynthesis.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and accessed as a PDF.