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

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). In enzymatic intermediate is very likely to contain the hyosamine [3-N- assays, HygK was found to interconvert UDP-glucose and methyl-2-DOS (4)] ring and the D-talose ring and is named UDP-galactose through C-4′ epimerization, and the reaction here 3-N-methyl-talamine (7)(Figure 2b). Connection of the reached an equilibrium favoring UDP-glucose after overnight destomic aldehyde ring to 7 would form 2. There are only two incubation (Figure S7). This balance may act to prevent glycosyltransferase-encoding genes within the hygromycin B depletion of the intracellular UDP-glucose pool. In contrast, biosynthetic gene cluster (Figure 2a): HygF belongs to the HygJ did not catalyze this interconversion. We tested whether GT-A fold superfamily and is likely to need a metal ion for its HygJ can epimerize 5 to talamine in vitro, but no new peak was function,28 while HygD belongs to the GT-B fold superfamily observed. Because 5 and talamine have the same MS and MS/ and needs no cofactor. These two candidates were cloned and MS spectra, these two species can hardly be distinguished if expressed in E. coli as recombinant proteins. Because HygF is they also have similar retention times. Therefore, pending annotated as a putative galactosyltransferase, an assay using further analysis, HygJ is still a candidate to catalyze UDP-galactose [the C-2′ epimer of UDP-talose (11)], 2-DOS epimerization, as the only other predicted epimerase encoded (3), and HygF with cofactor Mn2+ was performed. This in the cluster. incubation produced nearly complete conversion to a new The N-3 Methylation by HygM Occurs Independently + species at m/z 325.1618 ([C12H24N2O8 +H],calcd with the Formation of the Pseudodisaccharide. Meth- 325.1605) after 10 min (Figure 5a). Because no enzyme for ylation is a universal decoration in natural products that may isomerization was added, this species is thought to be significantly influence the biological activity and protect the galacamine (5)(Figure S2d). Another enzyme may epimerize molecule from destructive modification caused by resistance. A 5 to talamine, for eventual transformation to hygromycin B. unique putative methyltransferase-encoding gene hygM from Alternatively, given that HygF showed loose substrate the hyg cluster (Figure 2a) was cloned, expressed (Figure S5), specificity and accepted UDP-glucose with a low efficiency and tested in vitro. When using 3 and S-adenosylmethionine (Figure S6), it is possible that UDP-talose is the substrate for (SAM) as substrates, a new species at m/z 177.1234 + HygF, to generate the D-talose ring of talamine directly. In ([C7H16N2O3 +H], calcd 177.1234) was detected, indicating contrast, HygD showed no catalytic activity in this assay and is a single methylation (Figure 5b). With the structures of 2 and a strong candidate for being the enzyme responsible for adding 1 as a reference, the methylation site is most likely to be at N-3 the destomic aldehyde ring. to give 4. To determine the positions of HygM and HygF in

2206 DOI: 10.1021/acschembio.8b00375 ACS Chem. Biol. 2018, 13, 2203−2210 ACS Chemical Biology Articles the pathway, sequential reactions were performed, in which flour, 2% D-mannitol, and 2% agar) and TSBY liquid medium (3% one enzyme was first added for reaction, followed by heating tryptone soya broth, 0.5% yeast extract, and 10.5% sucrose) for for enzyme inactivation, and then the other was supplied for chromosomal DNA isolation and preparation of mycelium, respectively. E. coli strains were maintained in 2× TY medium (1% further catalysis. Surprisingly, 3-N-methyl-galacamine (6) ° (Figure S2e) was obtained in both assays, which showed that yeast extract, 1.6% tryptone, and 0.5% NaCl) at 37 C with appropriate antibiotic selection at final concentration of 100 μg/mL N-3 methylation and disaccharide formation are relatively ampicillin, 25 μg/mL chloramphenicol, and 50 μg/mL kanamycin. independent of each other in vitro (Figure 5c,d). This result Construction of the hygX Disruption Plasmid and an In- suggested that demethyl homologues of hygromycin B might Frame Deletion Mutant. To construct a plasmid for in-frame be formed. Accordingly, 3-N-demethyl-hygromycin C (8) deletion of hygX, two DNA fragments flanking hygX were amplified (Figure S2f)intheΔhygX strain and 3-N-demethyl- from the genomic DNA of DSM 40578 by using primers hygX-L1/L2 hygromycin B (9)(Figure S2g) in the wild-type strain were and hygX-R1/R2 (Table S5). The PCR products were cloned into − 33 34 detected via LC−ESI-HRMS and verified by MS/MS. Streptomyces E. coli shuttle vector pYH7 by Gibson assembly to Moreover, 8 was also converted to 9 by HygX in vitro under obtain gene disruption plasmid pWHU2847 (Table S1), which was verified by restriction endonuclease digestion and sequencing. To the same conditions under which 2 gave 1 (Figure 3c). create in-frame deletion mutant ΔhygX, the corresponding plasmid Therefore, intermediates lacking N-3 methylation are fully pWHU2847 was introduced into DSM 40578 by conjugation on a 9 ° processed to 3-N-demethyl-hygromycin B ( )(Figure 2b), just SFM plate (containing 100 mM CaCl2). After incubation at 28 C for as gentamicins lacking C-6′ methylation are also fully 14 h, the plate was overlaid with apramycin (25 μg/mL) and nalidixic processed.24 To verify the function of glycosyltransferase acid (25 μg/mL). Single apramycin-resistant exconjugants from this hygF and methyltransferase hygM in vivo, we repeatedly plate were patched on a SFM plate containing apramycin (25 μg/mL) attempted to delete them in frame but failed to obtain these and nalidixic acid (25 μg/mL) and grown at 28 °C for 2 or 3 days. To mutants because of the extreme intractability of the DSM screen the double-crossover mutant, each single colony from the 29 resistance double-check plate was patched onto SFM plates without 40578 strain. μ Sedoheptulose 7-phosphate, which is involved in the or with 25 g/mL apramycin. Genomic DNA of single apramycin- 30−32 sensitive colonies was extracted and checked by PCR using checking biosynthesis of lipopolysaccharide, may serve as the primers hygX-PC1 and hygX-PC2 (Table S5) and further confirmed origin of the UDP-destomic acid moiety of hygromycin B. by sequencing and Southern blot analysis (Figure S1). Recently, the biosynthesis of an intermediate, D-glycero-D-altro- Gene Complementation of the ΔhygX Mutant. For heptose 7-phosphate, was demonstrated by enzymatic assays, complementation, pWHU2848 and pWHU2849 (Table S1) were 35 and its following putative transformation to NDP-D-glycero-D- constructed by inserting hygX into vector pWHU77 under the altro-heptose was also predicted.29 For further decoration, control of constitutive promoter PermE* and the native promoter, 34 epimerization may be introduced and the amino group at the respectively, by Gibson assembly. After the sequence had been ′′ confirmed, the plasmid was introduced into ΔhygX by conjugation. C-6 site is thought to be installed through successive fi dehydrogenation and transamination to form the NDP- The complementary exconjugant was veri ed on SFM medium containing thiostrepton (25 μg/mL) and confirmed by PCR with destomic aldehyde moiety for linkage to the pseudodisacchar- fi checking primers hygX-CPC1 and hygX-CPC2 (Table S5). ide, accompanied by speci c epimerization, and optional Production, Extraction, and Analysis of Hygromycin B and methylation by HygX would then close the orthoester ring to Related Intermediates. For fermentation and detection of give the final products. hygromycin B and related intermediates, DSM 40578 and its mutant In summary, we have characterized the unusual orthoester were fermented in two stages. A seed culture was maintained in TSBY linkage formation performed by AKG/Fe(II)-dependent liquid medium at 28 °C while being shaken at 220 rpm for 36 h oxygenase HygX in vivo and in vitro and identified the elusive before being inoculated into liquid ISP2 medium (0.4% yeast extract, 1% malt extract, 0.4% glucose, and 1% inoculum) and then incubated direct precursor of hygromycin B. The independent formation ° of the pseudodisaccharide moiety and of N-3 methylation is at 28 C while being shaken at 220 rpm for 5 days. The fermentation broths were adjusted to pH 2.0 with H2SO4 and then agitated for 1 h. also described in vitro, which implies alternative pathways in fi fi fi The clari ed supernatant after centrifugation was ltered through hygromycin B biosynthesis. These ndings expand our Whatman filter paper and agitated with DOWEX 50 WX8-200 ion- knowledge of oxidative cyclization performed by enzymes exchange resin (1 g for 40 mL of broth) that was preconditioned with and of the biosynthesis of aminoglycoside antibiotics, a acetonitrile followed by Milli-Q water three times. After 1 h, the resin prerequisite for confident application of synthetic biology to was placed in a column, washed with Milli-Q water (6 column the engineering of these pathways. volumes), and eluted with 1 M NH4OH (6 column volumes). The eluate was freeze-dried, redissolved in Milli-Q water (0.2 mL of a concentrated solution was equivalent to 40 mL of broth), and filtered ■ METHODS μ through a 0.22 m microporous membrane before being subjected to Bacterial Strains, Chemicals, and Culture Conditions. E. coli LC−ESI-HRMS analysis. LC−ESI-HRMS analysis of extracts was DH10B was used as cloning host and E. coli ET12567/pUZ8002 for performed on a Thermo Electron LTQ-Orbitrap XL instrument fitted intergeneric conjugation between E. coli and Streptomyces. The S. with a Phenomenex Luna C18 column (250 mm × 4.6 mm) at a flow hygroscopicus subsp. hygroscopicus DSM 40578 wild-type strain was rate of 0.4 mL/min using a mobile phase of (A) 0.2% trifluoroacetic acquired from the China General Microbiological Culture Collection acid (TFA) in H2O (adjusted to pH 2.0 with NH4OH) and (B) 100% Center (CGMCC). Restriction endonucleases, Phusion High-Fidelity CH3CN. The following gradient was used for the separation of Master Mix with GC buffer, and Gibson Assembly Master Mix were hygromycin B and intermediates: 2% B from 0 to 2 min, 2 to 6% B obtained from New England Biolabs. Oligonucleotide primers were from 2 to 13 min, 6 to 90% B from 13 to 13.5 min, 90% B from 13.5 synthesized by GenScript and Tsingke. DNA sequencing of to 18 min, 90 to 2% B from 18 to 18.5 min, and 2% B from 18.5 to 24 polymerase chain reaction (PCR) products was performed by min. MS/MS analyses were performed in positive ionization mode GenScript or Tsingke. DIG DNA labeling and detection kits were with a 35% relative collision energy. purchased from Roche. Hygromycin B and 2-deoxystreptamine Isolation and Purification of Hygromycin C. Isolation of dihydrobromide were purchased from Sigma-Aldrich, and uridine hygromycin C from crude extract was performed on a Thermo 5′-diphosphogalactose disodium was from Coolaber. Wild-type DSM Scientific HPLC instrument (UltiMate 3000) fitted with an ELSD 40578 and its mutants were grown in SFM solid medium (3% soya (Alltech 2000ES) and a Phenomenex Synergi C18 column (250 mm

2207 DOI: 10.1021/acschembio.8b00375 ACS Chem. Biol. 2018, 13, 2203−2210 ACS Chemical Biology Articles

× 10 mm) at a flow rate of 3 mL/min using a mobile phase of (A) precipitate protein and filtered through a 0.22 μm microporous fl − 0.2% tri uoroacetic acid in H2O and (B) 100% CH3CN. The membrane for LC ESI-HRMS analysis. The boiled inactivation following gradient was used for the separation: 2 to 5% B from 0 to 6 protein was used as a negative control. min, 5 to 90% B from 6 to 7 min, 90% B from 7 to 11 min, 90 to 2% B The activity of HygX was determined by following the hygromycin from 11 to 12 min, and 2% B from 12 to 16 min. The temperature C consumption using HPLC−ELSD analysis. The reaction mixture and gas flow rate of the ELSD were set to 109 °C and 2.9 L/min, (1000 μL) for the assay of HygX activity contained enzyme (2.5 μM), respectively. α-ketoglutarate (2 mM), and various concentrations of hygromycin C NMR Characterization of Hygromycin C. For structure (0.075, 0.150, 0.375, 0.6, and 1.3 mM) in 25 mM Tris-HCl buffer elucidation of hygromycin C, one-dimensional (1H, 13C, and (pH 7.4) with 75 mM NaCl. The reaction mixture was incubated at DEPT) and two-dimensional (1H−1H COSY, HSQC, HMBC, 28 °C for 10 min. Samples (100 μL) were withdrawn every 2 min and HSQC-TOCSY, and NOESY) NMR spectra were recorded on an extracted with equal volumes of chloroform, which was filtered Agilent-NMR-vnmrs 600 spectrometer. Chemical shifts are reported through a 0.22 μm microporous membrane before HPLC−ELSD in parts per million using tetramethylsilane as an internal reference, analysis. Each experiment was repeated three times. and NMR data were processed using MestReNova software. Enzymatic Assay of HygM. The in vitro enzymatic assay of Construction of Protein Expression Plasmids, Overexpres- HygM was performed by combining 25 mM Tris-HCl (pH 7.4), 75 sion, and Purification of Proteins. Target genes hygX, hygM, hygJ, mM NaCl, 0.16 mM S-adenosylmethionine (SAM), 0.1 mM 2-DOS hygK, hygD, and hygF were amplified from the genomic DNA of DSM (3), and 3.5 μM purified recombinant HygM in a 200 μL total 40578 by PCR using corresponding primers (Table S5) with 30 cycles reaction volume. The reaction mixture was incubated at 28 °C for 3 h of denaturation at 98 °C for 30 s, annealing at 58 °C for 30 s, and followed by treatment with an equal volume of chloroform to extension at 72 °C for 30 s per kilobase plus final extension at 72 °C precipitate protein and then filtered through a 0.22 μm microporous for 10 min. The PCR product was purified by gel extraction and membrane before being subjected to LC−ESI-HRMS analysis. The inserted into vector pET28a(+) between NdeI and EcoRI to create boiled inactivation HygM was used as a negative control. To protein expression plasmids pWHU2856, pWHU2857, pWHU2858, determine the reaction order of HygM and HygF, the HygM assay pWHU2859, pWHU2860, and pWHU2861 (Table S1). The resulting described above was abolished in boiling water and then 0.6 μM constructs were verified by restriction endonuclease digestion and HygF was added with 50 μMMn2+ and 0.1 mM UDP-galactose for DNA sequencing and then used to transform E. coli BL21(DE3). The further incubation at 28 °C for 3 h and LC−ESI-HRMS analysis. transformant was grown at 37 °C in LB medium (0.5% yeast extract, Enzymatic Assay of HygF. The in vitro enzymatic assay of HygF 1% tryptone, 1% NaCl, and 50 μg/mL kanamycin) while being was performed by combining 25 mM Tris-HCl (pH 7.4), 75 mM − μ shaken until the OD600 reached 0.4 0.6. After addition of 0.1 mM NaCl, 50 M MnCl2, 0.1 mM 2-DOS, 0.1 mM UDP-galactose, and isopropyl β-D-1-thiogalactopyranoside for induction, the cultures were 0.6 μM purified recombinant HygF in a 200 μL total reaction volume. shaken at 18 °C for 16 h, harvested by centrifugation at 5000 rpm for The reaction mixture was incubated at 28 °C for 3 h followed by 15 min, and resuspended in lysis buffer [150 mM NaCl and 50 mM treatment with an equal volume of chloroform to precipitate protein Tris-HCl (pH 7.8)]. Cells were disrupted by sonication (30 min with and filtered through a 0.22 μm microporous membrane before being a 25% duty cycle), and the lysate was clarified by centrifugation subjected to LC−ESI-HRMS analysis. The boiled inactivation protein (12000 rpm and 4 °C for 1 h). The supernatant was passed through a was used as a negative control. To determine the reaction order of 5 mL IMAC column (GE Healthcare) charged with nickel and HygF and HygM, the HygF assay described above was abolished in previously equilibrated with lysis buffer. Proteins were eluted using a boiling water and then 3.5 μM HygM was added with 0.16 mM SAM linear gradient of imidazole (≤800 mM) in a buffer of 50 mM Tris- for further incubation at 28 °C for 3 h and LC−ESI-HRMS analysis. HCl (pH 7.8) and 150 mM NaCl. Fractions containing target Enzymatic Assays of HygJ and HygK. The in vitro enzymatic proteins were concentrated using Amicon-Ultra Centrifugal Filters assay of HygJ or HygK was performed by combining 25 mM Tris-HCl (Millipore) and further purified using a PD-10 column (GE (pH 7.4), 75 mM NaCl, 0.4 mM NAD+/NADP+, 0.2 mM UDP- Healthcare) equilibrated in 50 mM Tris-HCl (pH 7.8) and 150 glucose/UDP-galactose, and 0.6 μM purified recombinant HygJ/ mM NaCl. Fractions containing HygX, HygM, HygJ, HygK, HygD, HygK in a 50 μL total reaction volume. The reaction mixture was and HygF proteins were concentrated to 32, 2, 3, 1, 17, and 19 mg incubated at 28 °C overnight followed by treatment with an equal mL−1, respectively, and stored in 10% glycerol at −80 °C until they volume of chloroform. The boiled inactivation protein was used as a were used. All these proteins were confirmed by sodium dodecyl negative control. The analysis of UDP-hexose as a substrate or sulfate−polyacrylamide gel electrophoresis. product was performed via HPLC connected to a SPD-M20A Identification of Proteins. The exact masses of proteins HygX, ultraviolet absorption detector (Shimadzu) fitted with a COSMOSIL HygM, HygJ, HygK, HygD, and HygF without methionine were PBr column (250 mm × 4.6 mm) at a flow rate of 0.8 mL/min. The verified by mass spectra on a Thermo Electron LTQ-Orbitrap XL mobile phase was a sodium phosphate solution (100 mM, pH 7.0). fitted with a Phenomenex Jupiter C4 column (250 mm × 2 mm) at a UDP-hexose showed significant absorption at a wavelength of 254 flow rate of 0.3 mL/min using a mobile phase of (A) 0.1% TFA in nm. H2O and (B) 0.1% TFA in CH3CN. The following gradient was used for the separation of protein: 5% B from 0 to 1 min, 5 to 95% B from ■ ASSOCIATED CONTENT 1 to 20 min, 95% B from 20 to 25 min, 95 to 5% B from 25 to 27 min, *S Supporting Information and 5% B from 27 to 30 min. The mass spectrometer was set to full The Supporting Information is available free of charge on the scan (from m/z 300 to 2000). The mass spectrometric data were ACS Publications website at DOI: 10.1021/acschem- processed and deconvoluted using the Bioworks software (Thermo Finnigan). bio.8b00375. Enzymatic Assay and Kinetic Characterization of HygX. The Tables S1−S5 and Figures S1−S7 (PDF) in vitro enzymatic assay of HygX under open air conditions or in an anaerobic chamber (Coy Laboratories) was performed by combining 2+ ■ AUTHOR INFORMATION 25 mM Tris-HCl (pH 7.4), 75 mM NaCl, and 0.1 mM Fe with 10 μM purified recombinant HygX for preincubation for 15 min, Corresponding Author followed by the addition of 0.5 mM α-ketoglutarate and 0.15 mM *E-mail: [email protected]. μ hygromycin C in a 200 L total reaction volume. For the assay to ORCID convert 3-N-demethyl-hygromycin C (8)to3-N-demethyl-hygromycin B (9), the fermentation broth of ΔhygX was applied as a Yuhui Sun: 0000-0002-9258-2639 ° Author Contributions substrate. The reaction mixture was typically incubated at 28 C for 3 † h followed by treatment with an equal volume of chloroform to S.L. and J.Z. contributed equally to this work.

2208 DOI: 10.1021/acschembio.8b00375 ACS Chem. Biol. 2018, 13, 2203−2210 ACS Chemical Biology Articles

Notes (16) Arenz, S., Juette, M. F., Graf, M., Nguyen, F., Huter, P., The authors declare no competing ﬁnancial interest. Polikanov, Y. S., Blanchard, S. C., and Wilson, D. N. (2016) Structures of the orthosomycin antibiotics avilamycin and evernimicin ■ ACKNOWLEDGMENTS in complex with the bacterial 70S ribosome. Proc. Natl. Acad. Sci. U. S. A. 113, 7527−7532. This work was supported by the National Natural Science (17) Tang, M. C., Zou, Y., Watanabe, K., Walsh, C. T., and Tang, Y. Foundation of China (31470186). The authors thank Z. Ding (2017) Oxidative cyclization in natural product biosynthesis. Chem. (Yunnan University, Kunming, China) for his help in NMR Rev. 117, 5226−5333. analysis of hygromycin C and P. F. Leadlay (University of (18) McCulloch, K. M., McCranie, E. K., Smith, J. A., Sarwar, M., Cambridge, Cambridge, U.K.) for his critical reading of the Mathieu, J. L., Gitschlag, B. L., Du, Y., Bachmann, B. O., and Iverson, manuscript. T. M. (2015) Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily. Proc. Natl. ■ REFERENCES Acad. Sci. U. S. A. 112, 11547−11552. (19) Wang, C., Chang, W. C., Guo, Y., Huang, H., Peck, S. C., (1) Mann, R. L., and Bromer, W. W. (1958) The isolation of a Pandelia, M. E., Lin, G. M., Liu, H. W., Krebs, C., and Bollinger, J. M. second antibiotic from Streptomyces hygroscopicus. J. Am. Chem. Soc. − (2013) Evidence that the fosfomycin-producing epoxidase, HppE, is a 80, 2714 2716. − (2) Cameron, A., and Gaynor, E. C. (2014) Hygromycin B and non-heme-iron peroxidase. Science 342, 991 995. (20) Strieker, M., Kopp, F., Mahlert, C., Essen, L. O., and Marahiel, apramycin antibiotic resistance cassettes for use in Campylobacter β jejuni. PLoS One 9, e95084. M. A. (2007) Mechanistic and structural basis of stereospecific c - hydroxylation in calcium-dependent antibiotic, a daptomycin-type (3) Radman, I., Greiss, S., and Chin, J. W. (2013) Efficient and rapid − C. elegans transgenesis by bombardment and hygromycin B selection. lipopeptide. ACS Chem. Biol. 2, 187 196. PLoS One 8, e76019. (21) Blasiak, L. C., Vaillancourt, F. H., Walsh, C. T., and Drennan, C. L. (2006) Crystal structure of the non-haem iron halogenase SyrB2 (4) Blochlinger, K., and Diggelmann, H. (1984) Hygromycin B − phosphotransferase as a selectable marker for DNA transfer in syringomycin biosynthesis. Nature 440, 368 371. experiments with higher eucaryotic cells. Mol. Cell. Biol. 4, 2929− (22) Zhang, Z., Ren, J., Stammers, D. K., Baldwin, J. E., Harlos, K., 2931. and Schofield, C. J. (2000) Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase. Nat. Struct. Biol. 7, (5) Macintyre, G., Woods, D. E., and Anderson, R. (1991) − Hygromycin B inhibits synthesis of murine coronavirus RNA. 127 133. Antimicrob. Agents Chemother. 35, 2630−2633. (23) Komaki, H., Ichikawa, N., Oguchi, A., Hamada, M., Tamura, T., (6) Pfister, P., Risch, M., Brodersen, D. E., and Böttger, E. C. (2003) Fujita, N., and Suzuki, K. I. (2017) Genome analysis-based Role of 16S rRNA helix 44 in ribosomal resistance to hygromycin B. reclassification of Streptomyces endus and Streptomyces sporocinereus − as later heterotypic synonyms of Streptomyces hygroscopicus subsp. Antimicrob. Agents Chemother. 47, 1496 1502. − (7) McGaha, S. M., and Champney, W. S. (2007) Hygromycin B hygroscopicus. Int. J. Syst. Evol. Microbiol. 67, 343 345. inhibition of protein synthesis and ribosome biogenesis in Escherichia (24) Guo, J., Huang, F., Huang, C., Duan, X., Jian, X., Leeper, F., coli. Antimicrob. Agents Chemother. 51, 591−596. Deng, Z., Leadlay, P. F., and Sun, Y. (2014) Specificity and (8) Eustice, D. C., and Wilhelm, J. M. (1984) Mechanisms of action promiscuity at the branch point in gentamicin biosynthesis. Chem. − of aminoglycoside antibiotics in eucaryotic protein synthesis. Biol. 21, 608 618. Antimicrob. Agents Chemother. 26,53−60. (25) Kovaleva, E. G., and Lipscomb, J. D. (2008) Versatility of (9) Carter, A. P., Clemons, W. M., Brodersen, D. E., Morgan- biological non-heme Fe(II) centers in oxygen activation reactions. − Warren, R. J., Wimberly, B. T., and Ramakrishnan, V. (2000) Nat. Chem. Biol. 4, 186 193. Functional insights from the structure of the 30S ribosomal subunit (26) Wu, L. F., Meng, S., and Tang, G. L. (2016) Ferrous iron and α and its interactions with antibiotics. Nature 407, 340−348. -ketoglutarate-dependent dioxygenases in the biosynthesis of (10) Borovinskaya, M. A., Shoji, S., Fredrick, K., and Cate, J. H. D. microbial natural products. Biochim. Biophys. Acta, Proteins Proteomics − (2008) Structural basis for hygromycin b inhibition of protein 1864, 453 470. biosynthesis. RNA 14, 1590−1599. (27) Grzyska, P. K., Ryle, M. J., Monterosso, G. R., Liu, J., Ballou, D. (11) Kudo, F., Yamamoto, Y., Yokoyama, K., Eguchi, T., and P., and Hausinger, R. P. (2005) Steady-state and transient kinetic Kakinuma, K. (2005) Biosynthesis of 2-deoxystreptamine by three analyses of taurine/alpha-ketoglutarate dioxygenase: effects of oxygen crucial enzymes in Sreptomyces fradiae NBRC 12773. J. Antibiot. 58, concentration, alternative sulfonates, and active-site variants on the 766−774. FeIV-oxo intermediate. Biochemistry 44, 3845−3855. (12) Weitnauer, G., Mühlenweg, A., Trefzer, A., Hoffmeister, D., (28) Hu, Y., and Walker, S. (2002) Remarkable structural similarities Süßmuth, R. D., Jung, G., Welzel, K., Vente, A., Girreser, U., and between diverse glycosyltransferases. Chem. Biol. 9, 1287−1296. Bechthold, A. (2001) Biosynthesis of the orthosomycin antibiotic (29) Tang, W., Guo, Z., Cao, Z., Wang, M., Li, P., Meng, X., Zhao, avilamycin A: deductions from the molecular analysis of the avi X., Xie, Z., Wang, W., Zhou, A., Lou, C., and Chen, Y. (2018) d- biosynthetic gene cluster of Streptomyces viridochromogenes Tü57 and sedoheptulose-7-phosphate is a common precursor for the heptoses of production of new antibiotics. Chem. Biol. 8, 569−581. septacidin and hygromycin B. Proc. Natl. Acad. Sci. U. S. A. 115, (13) Weitnauer, G., Gaisser, S., Kellenberger, L., Leadlay, P. F., and 2818−2823. Bechthold, A. (2002) Analysis of a C-methyltransferase gene (aviG1) (30) Taylor, P. L., Blakely, K. M., De Leon, G. P., Walker, J. R., involved in avilamycin biosynthasesis in Streptomyces viridochromo- McArthur, F., Evdokimova, E., Zhang, K., Valvano, M. A., Wright, G. genes Tü57 and complementation of a Saccharopolyspora erythraea D., and Junop, M. S. (2008) Structure and function of sedoheptulose- eryBIII mutant by aviG1. Microbiology 148, 373−379. 7-phosphate isomerase, a critical enzyme for lipopolysaccharide (14) Hofmann, C., Boll, R., Heitmann, B., Hauser, G., Dürr, C., biosynthesis and a target for antibiotic adjuvants. J. Biol. Chem. 283, Frerich, A., Weitnauer, G., Glaser, S. J., and Bechthold, A. (2005) 2835−2845. Genes encoding enzymes responsible for biosynthesis of L-Lyxose and (31) Valvano, M. A., Messner, P., and Kosma, P. (2002) Novel attachment of eurekanate during avilamycin biosynthesis. Chem. Biol. pathways for biosynthesis of nucleotide-activated glycero-manno- 12, 1137−1143. heptose precursors of bacterial glycoproteins and cell surface (15) Vey, J. L., Al-Mestarihi, A., Hu, Y., Funk, M. A., Bachmann, B. polysaccharides. Microbiology 148, 1979−1989. O., and Iverson, T. M. (2010) Structure and mechanism of orf 36,an (32) Yu, C. K., Wang, C. J., Chew, Y., Wang, P. C., Yin, H. S., and amino sugar oxidizing enzyme in everninomicin biosynthesis. Kao, M. C. (2016) Functional characterization of helicobacter pylori Biochemistry 49, 9306−9317. 26695 sedoheptulose 7-phosphate isomerase encoded by hp0857 and

2209 DOI: 10.1021/acschembio.8b00375 ACS Chem. Biol. 2018, 13, 2203−2210 ACS Chemical Biology Articles its association with lipopolysaccharide biosynthesis and adhesion. Biochem. Biophys. Res. Commun. 477, 794−800. (33) Sun, Y., He, X., Liang, J., Zhou, X., and Deng, Z. (2009) Analysis of functions in plasmid pHZ1358 influencing its genetic and structural stability in Streptomyces lividans 1326. Appl. Microbiol. Biotechnol. 82, 303−310. (34) Gibson, D. G., Benders, G. A., Andrews-Pfannkoch, C., Denisova, E. A., Baden-Tillson, H., Zaveri, J., Stockwell, T. B., Brownley, A., Thomas, D. W., Algire, M. A., Merryman, C., Young, L., Noskov, V. N., Glass, J. I., Venter, J. C., Hutchison, C. A., and Smith, H. O. (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215−1220. (35) Huang, C., Huang, F., Moison, E., Guo, J., Jian, X., Duan, X., Deng, Z., Leadlay, P. F., and Sun, Y. (2015) Delineating the biosynthesis of gentamicin X2, the common precursor of the gentamicin C antibiotic complex. Chem. Biol. 22, 251−261.

2210 DOI: 10.1021/acschembio.8b00375 ACS Chem. Biol. 2018, 13, 2203−2210