Kinases are annotated in many nucleoside biosynthetic gene clusters but generally are considered responsible only for self-resistance. Here, we report an unexpected 2′-phosphorylation of nucleoside biosynthetic intermediates in the nikkomycin and polyoxin pathways. This phosphorylation is a unique cryptic modification as it is introduced in the third of seven steps during aminohexuronic acid (AHA) nucleoside biosynthesis, retained throughout the pathway’s duration, and is removed in the last step of the pathway. Bioinformatic analysis of reported nucleoside biosynthetic gene clusters indicates the presence of cryptic phosphorylation in other pathways and the importance of functional characterization of kinases in nucleoside biosynthetic pathways in general. This study also functionally characterized all of the enzymes responsible for AHA biosynthesis and revealed that AHA is constructed via a unique oxidative C–C bond cleavage reaction. The results indicate a divergent biosynthetic mechanism for three classes of antifungal nucleoside natural products.
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The authors declare that all the data supporting the findings of this study are available within the paper, Extended Data and Supplementary Information. All plasmids, analytical amounts of reported compounds, and raw data are available upon request. Sequences are deposited at National Center for Biotechnology Information under accession nos.: PolQ2, ABX24486; PolJ, ABX24494; PolK, ABX24493; PolD, ABX24500; NikL, CAC80910; NikM, CAC80911; NikK, CAC80909; NikS, CAC11141.
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We thank the Duke University Shared Instruments Core Facility (P. Silinski) and the Duke University Lipidomics Core Facility (Z. Guan) for assistance with the LC–HRMS and LC–HRMS/MS analyses, respectively. We thank the Duke University NMR Spectroscopy Core Facility (B. Bobay, R. Venters and D. Mika) for their assistance with the NMR analyses. We thank A. Stelling for assistance with the FTIR analyses. This work was supported by the Duke University School of Medicine, and National Institute of General Medical Sciences R01 GM115729 (to K.Y.). M.M.D. was supported in part by the Duke Medical Scientist Training Program (T32 GM007171).
The authors declare no competing interests.
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Shown are HPLC chromatograms at 260 nm for a full reaction with PolK, a full reaction with PolD, a control without enzyme, and a control without α-KG. No consumption of 5′-OAP was detected. The results were reproducible in at least two independent assays.
Shown are HPAEC chromatograms at 260 nm for a control without the enzyme (trace i) and the complete reaction (trace ii). No consumption of EP-UMP is detected. The results were reproducible in at least two independent assays.
Shown are HPAEC chromatograms at 260 nm for a control without the enzyme (trace i), an assay with PolD (ii), an assay with PolK (iii), and an assay with NikM (iv). No consumption of OABP was observed after 24 hours. The results were reproducible in at least two independent assays.
a. PolJ phosphatase (10 μM) was incubated with 2′-HAP (100 μM) in 50 mM Tris pH 8.0 supplemented with 10 mM MgCl2 at 25 °C for 18 hrs. b. NikK aminotransferase (30 μM) was incubated with 2′-HAP (200 μM) in 200 mM Tris pH 9.0 supplemented with 1 mM MgCl2 and 10 mM l-Glu at 25 °C for 2 hrs. c. PolK oxygenase (25 μM) was incubated with 2′-HAP (200 μM) in 150 mM NaCl, 50 mM Tris pH 7.5 supplemented with 1 mM Fe2+, 1 mM ascorbate, 200 μM α-KG at 25 °C for 18 hrs. No product formation was detected. The results were reproducible in at least two independent assays.
PolK (40 μM) was incubated with KOAP (100 μM) in the presence of 100 μM Fe2+, 2 mM ascorbate, and 1 mM α-KG for 20 hours at 25 °C. Even after prolonged incubation (20 hours), no product is observed. The results were reproducible in at least two independent assays.
PolD (40 μM) was incubated with KOAP (100 μM) in the presence of 100 μM Fe(II), 1 mM ascorbate, and 2 mM α-KG for 2 hours at 25 °C. No product formation or KOAP consumption. For comparison, under similar conditions, PolD completed the conversion of AHOAP to AHAP in <15 minutes. The results were reproducible in at least two independent assays.
Coupling constants for possible stereochemistry of C4′, C5′, C6′ and C7′. Experimental evidence of JH3′-H4′ = 10.7 Hz, JH4′-H5′ = 3.5 Hz, JH5′-H6′ = 3.5 Hz and JH6′-H7′ = 2.1 Hz for AHOA is most consistent with the stereochemistry of 4′R, 5′R, 6′R, 7′S, indicating that AHOA and AHOAP have 4′R, 5′R, 6′R, 7′S stereochemistry. Dihedral angles were calculated with ChemDraw Professional v19.0 and ChemDraw 3D v19.0 (PerkinElmer Informatics).
The PolD assays with AHOAP were performed with 10 μM PolD, 0.1mM Fe2+, 2 mM ascorbate, 100 μM AHOAP in 150 mM NaCl, 50 mM Tris pH 7.6, 10% glycerol. The PolD assays with 2′-OAP were performed with 30μM PolD, 1mM ascorbate, 0.5 mM Fe2+, 200 μM 2’-OAP in 150 mM NaCl, 50 mM Tris pH 7.6.
Shown are EICs (calculated m/z for [M+H]+ ± 5 ppm) for polyoxins A, B, D, F, G, H, I, and J. No polyoxin production was detected in ΔpolQ2 (a), polyoxin A, B, F, and G were found in the wt strain (b), and polyoxin A and F were detected in the ΔpolQ2 + polQ2 strain (c). The observations were reproducible for 2-3 different clones for each mutant strain. The culture was repeated twice for each clone.
Proposed tailoring of sugar size by oxidative C-C bond cleavage by PolD homologs.
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Draelos, M.M., Thanapipatsiri, A., Sucipto, H. et al. Cryptic phosphorylation in nucleoside natural product biosynthesis. Nat Chem Biol 17, 213–221 (2021). https://doi.org/10.1038/s41589-020-00656-8
Microbial Cell Factories (2022)
Blocks in the pseudouridimycin pathway unlock hidden metabolites in the Streptomyces producer strain
Scientific Reports (2021)