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Direct Genetic and Enzymatic Evidence for Oxidative Cyclization in Hygromycin B Biosynthesis

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Direct Genetic and Enzymatic Evidence for Oxidative Cyclization in Hygromycin B Biosynthesis Articles Cite This: ACS Chem. Biol. 2018, 13, 2203−2210 Direct Genetic and Enzymatic Evidence for Oxidative Cyclization in Hygromycin B Biosynthesis † † Sicong Li, Jun Zhang, Yuanzhen Liu, Guo Sun, Zixin Deng, and Yuhui Sun* Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, People’s Republic of China *S Supporting Information ABSTRACT: Hygromycin B is an aminoglycoside antibiotic with a structurally distinctive orthoester linkage. Despite its long history of use in industry and in the laboratory, its biosynthesis remains poorly understood. We show here, by in-frame gene deletion in vivo and detailed enzyme characterization in vitro, that formation of the unique orthoester moiety is catalyzed by the α-ketoglutarate- and non-heme iron-dependent oxygenase HygX. In addition, we identify HygF as a glycosyltransferase adding UDP-hexose to 2-deoxystreptamine, HygM as a methyltransferase responsible for N-3 methylation, and HygK as an epimerase. These experimental results and bioinformatic analyses allow a detailed pathway for hygromycin B biosynthesis to be proposed, including the key oxidative cyclization reactions. ygromycin B is an aminoglycoside antibiotic produced epoxidation,20 desaturation,21 and halogenation22 via radical H by Streptomyces hygroscopicus.1 Since its discovery in the intermediates,17 are responsible for the formation of the 1950s, it has become widely used as a veterinary drug to orthoester linkage. However, no authentic uncyclized precursor control infections of intestinal parasites in chickens and swine. of orthoesters has been isolated so far, and no direct In biological studies, hygromycin B also serves as a useful biochemical evidence for their function has been presented. selection agent in both bacteria2 and eukaryotic cells.3,4 Moreover, its antiviral activity in vivo and in vitro was also ■ RESULTS AND DISCUSSION Downloaded via WUHAN UNIV on September 26, 2018 at 01:04:15 (UTC). 5 reported. Hygromycin B targets the bacterial 30S ribosomal HygX Is Responsible for Orthoester Linkage For- subunit6,7 and eukaryotic ribosomes8 to perturb protein 9 mation in Hygromycin B Biosynthesis As Proven in synthesis. In particular, it potently inhibits spontaneous Vivo. 10 On the basis of previous analysis of the hygromycin B See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. reverse translocation. However, detailed knowledge of its biosynthetic gene cluster (GenBank accession number biosynthesis has been mainly limited to the assembly of its 2- AJ628642.1) (Figure 2a), the predicted oxidase HygX is the 11 deoxystreptamine (2-DOS) core, which is common to many most promising candidate for catalysis of orthoester aminoglycosides. Notably, hygromycin B contains a unique formation.18 We therefore aimed to remove the hygX gene orthoester moiety (Figure 1). This spirocyclic ortho-δ-lactone from the hygromycin B-producing strain Streptomyces hygro- confers special a structural property on antibiotics such as scopicus subsp. hygroscopicus DSM 4057823 by in-frame − avilamycin12 14 and everninomicin,15,16 and in hygromycin B, deletion (Figure S1). However, when we attempted to it serves as the linkage between the D-talose ring and the approach it through conjugation, we encountered tremendous destomic acid ring. Until now, the study of orthoester difficulty in achieving exconjugants and the double-crossover biosynthesis has lagged behind that of other enzyme-induced mutant. We finally obtained the ΔhygX strain after several oxidative cyclizations17 and has mainly been performed at the months of repeated experiments. Analysis by liquid chroma- structural level.18 For example, on the basis of the crystal tography coupled with electrospray ionization high-resolution structure and measurements of binding affinity with the mass spectrometry (LC−ESI-HRMS) showed the presence of cyclized product, it has been suggested that a set of α- ketoglutarate, non-heme iron-dependent [AKG/Fe(II)-de- Received: April 23, 2018 pendent] enzymes, which have been reported to catalyze Accepted: June 7, 2018 inter alia oxidative cyclization, hydroxylation, peroxidation,19 Published: June 7, 2018 © 2018 American Chemical Society 2203 DOI: 10.1021/acschembio.8b00375 ACS Chem. Biol. 2018, 13, 2203−2210 ACS Chemical Biology Articles Figure 1. Representative natural products containing orthoester linkages. The orthoester linkages in each compound are colored red. + hygromycin B (1)atm/z 528.2392 ([C20H37N3O13 +H], frequently seen as intermediates and shunt metabolites in other calcd 528.2399) in the wild type (Figure S2a), but it was aminoglycoside pathways.24 The structure of this newly completely abolished in ΔhygX (Figure 3a). To verify that this identified compound 2 was consistent with it being an was caused by the specific absence of hygX, wild-type hygX was intermediate and the substrate for cyclization. To test this, inserted into plasmid pWHU77 under the control of the recombinant HygX (Figure S5) was purified from Escherichia PermE* promoter and transferred into ΔhygX to generate coli and used for enzymatic assays in vitro. As a member of the ΔhygX::pWHU2848 (Tables S1 and S2). However, 1 was still AKG/Fe(II)-dependent enzyme family,25,26 HygX requires missing in this complementation strain (Figure 3a). Consid- Fe2+ as a cofactor.18 Therefore, it was preincubated with 0.1 ering that PermE* might not work in DSM 40578, we mM Fe2+ for 15 min followed by an overnight reaction with 2 constructed an alternative version of complementation plasmid and α-ketoglutarate. As shown, 2 was converted to 1 by HygX ΔhygX::pWHU2849 (Tables S1 and S2) housing a longer with a conversion rate of 95% (Figure 3b). This reaction did fragment containing hygX and a 81 bp sequence upstream of its not occur if either Fe2+ or α-ketoglutarate were absent. start codon, which should house the native promoter. In this Considering AKG/Fe(II)-dependent enzymes are generally case, the production of 1 was restored, as expected, to a level sensitive to oxidative damage18 that might be induced by similar to that of the wild type (Figure 3a). Therefore, HygX oxygen, HygX was preincubated with Fe2+ under anaerobic was confirmed to be involved in the biosynthesis but rigorously conditions before being exposed to air for catalysis. As not verified to be responsible for catalyzing the cyclization step. expected, the conversion rate under such conditions was AKG/Fe(II)-Dependent Enzyme HygX Converts Un- improved, especially when the reaction time was short (Table cyclized Hygromycin C to Hygromycin B in Vitro and Its S4). Enzymatic Characterization. Instead of hygromycin B, the Kinetic parameters of HygX were determined with purified 2 ΔhygX extract was found to contain a new compound at m/z as the substrate. High-performance liquid chromatography + − 530.2553 ([C20H39N3O13 +H], calcd 530.2556). It was with an evaporative light scattering detector (HPLC ELSD) verified as a ring-open metabolite by tandem mass spectrom- was used to monitor the disappearance of hygromycin C. The etry (MS/MS) and nuclear magnetic resonance (NMR) data were fitted to the integrated form of the Michaelis− (Figures S2b and S4 and Table S3) and is named here Menten equation using Origin version 9.0, and the calculated ± ± −1 hygromycin C (2)(Figure 2b). Compound 2 could also be KM for 2 was 0.14 0.02 mM and the kcat 11.04 0.00 s detected as a minor component in wild-type extracts, which is (Figure 4). The kinetic parameters of HygX are comparable to 2204 DOI: 10.1021/acschembio.8b00375 ACS Chem. Biol. 2018, 13, 2203−2210 ACS Chemical Biology Articles Figure 2. Biosynthetic gene cluster and proposed model for hygromycin B biosynthesis. (a) Organization of the hygromycin B biosynthetic gene cluster from DSM 40578. (b) Pathway of hygromycin B biosynthesis. Solid arrows indicate conversions confirmed by in vivo or/and in vitro studies. Dashed arrows refer to putative steps without experimental evidence. Molecule structures colored blue, red, and green are building blocks related to hyosamine, D-talose, and destomic acid rings of hygromycin B biosynthesis, respectively. those of TauD, another member of the AKG/Fe(II)- investigate earlier steps in the biosynthetic pathway of ± fi dependent enzyme family, for its substrate (KM =58 0.6 hygromycin B, we rst screened wild-type extracts for the μ ± −1 27 M, and kcat = 12.5 0.5 s ). presence of disaccharide intermediates. A compound at m/z + HygF Links 2-DOS and UDP-Galactose To Build the 339.1762 ([C13H26N2O8 +H], calcd 339.1762) was detected Pseudodisaccharide Scaffold of Hygromycin B. To with a fragment at m/z 177.04 by MS/MS (Figure S2c). 2205 DOI: 10.1021/acschembio.8b00375 ACS Chem. Biol. 2018, 13, 2203−2210 ACS Chemical Biology Articles Figure 3. Genetic and enzymatic analysis of orthoester moiety formation catalyzed by HygX. LC−ESI-HRMS analysis of (a) strain extracts, (b) in vitro conversion of hygromycin C (2) to hygromycin B (1), and (c) in vitro conversion of 3-N-demethyl-hygromycin C (8)to 3-N-demethyl-hygromycin B (9). Figure 5. LC−ESI-HRMS analysis of conversions by HygF and HygM in vitro. 2-DOS and UDP-galactose were used as substrates with (a) HygF, (b) HygM, (c) HygM followed by HygF, (d) HygF Figure 4. Kinetic constants of AKG/Fe(II)-dependent oxidase HygX. followed by HygM, and (e) no enzyme as a control. Blue, green, (a) Standard curve of hygromycin C, where lgC and lgA represent the yellow, and red traces indicate the extracted ion chromatograms of 2- natural logarithm of hygromycin C concentration (25−2000 μM) and DOS (3), hyosamine (4), galacamine (5), and 3-N-methyl-galacamine peak area, respectively. (b) Michaelis−Menten saturation curve of (6), respectively, in each panel. HygX. Error bars represent the standard deviation of the mean. There are two putative epimerases or dehydrogenases, HygJ and HygK, in the hyg cluster that might generate UDP- According to the structure of 2, the pseudodisaccharide galactose as the substrate for HygF (Figure 2).
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