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Shaotong Zhou
Shenyang Pharmaceutical University
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ORCiD: https://orcid.org/0000-0003-0340-8610
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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.
Keywords
gentamicin, C-3',4'-dideoxygenation, phosphotransferase, pyridoxal-5′- phosphate (PLP)-dependent enzyme
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- SI20201217.pdf
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
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See the published version of this preprint at Microbial Cell Factories.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Shaotong Zhou
Shenyang Pharmaceutical University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-0340-8610
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Microbial Cell Factories.
Version 1
Posted 22 Dec, 2020
No community comments so far
Editorial decision: Major Revision
On 31 Jan, 2021
Review #2 received
Received 26 Jan, 2021
Review #3 received
Received 26 Jan, 2021
Reviewer #3 agreed
On 10 Jan, 2021
Review #1 received
Received 09 Jan, 2021
Reviewer #2 agreed
On 04 Jan, 2021
Reviewer #1 agreed
On 25 Dec, 2020
Reviewers invited
Invitations sent on 20 Dec, 2020
Editor assigned
On 17 Dec, 2020
Submission checks complete
On 17 Dec, 2020
Editor invited
On 17 Dec, 2020
First submitted
On 16 Dec, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Microbiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Microbial Cell Factories.
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
This is a list of supplementary files associated with this preprint. Click to download.
- SI20201217.pdf
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
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