Direct Genetic and Enzymatic Evidence for Oxidative Cyclization in

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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 ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00375 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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ACS Chemical Biology

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.L. and J.Z. contributed equally to this work.

* To whom correspondence should be addressed. E-mail: [email protected]

1

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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. we identify HygF

as

a

glycosyltransferase

adding

Further,

UDP-hexose

to

2-deoxystreptamine, HygM as a methyltransferase responsible for N-3 methylation, and HygK as an epimerase. These experimental results and bioinformatic analysis allow a detailed pathway for hygromycin B biosynthesis to be proposed, including the key oxidative cyclization reactions.

2


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INTRODUCTION Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus1. Since its discovery in 1950s it has become widely used as a veterinary drug to control infections of intestinal parasites in chickens and swine. In biological studies, hygromycin B also serves as a useful selection agent in both bacteria2 and eukaryotic cells.3,4 Moreover, its antiviral activity in vivo and in vitro was also reported.5 Hygromycin B targets the bacterial 30S ribosomal-subunit6,7 and eukaryotic ribosomes8 to perturb protein synthesis.9 In particular, it potently inhibits spontaneous reverse translocation.10 However, detailed knowledge of its biosynthesis has been mainly limited to the assembly of its 2-deoxystreptamine (2-DOS) core,11 which is common to many aminoglycosides. Notably, hygromycin B contains a unique orthoester moiety (Figure 1). This spirocyclic ortho-δ-lactone confers special structural property on antibiotics such as avilamycin12-14 and everninomicin,15,16 and in hygromycin B it serves as the linkage between the

D-talose

ring and the

destomic acid ring. Until now, the study of orthoester biosynthesis has lagged behind that of other enzyme-induced oxidative cyclizations17 and has mainly been carried out at the structural level.18 For example, based on crystal structure and measurements of binding affinity with cyclized product it has been suggested that a set of α-ketoglutarate, non-heme iron-dependent (AKG/Fe(II)-dependent) enzymes, which have been reported to catalyze inter alia

oxidative

cyclization,

hydroxylation,

peroxidation,19

3


epoxidation,20


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desaturation,21 and halogenation22 via radical intermediates,17 are responsible for the formation of the orthoester linkage. However, no authentic uncyclized precursor of orthoesters has been isolated so far, and no direct biochemical evidence for their function has been presented.

RESULTS AND DISCUSSION HygX is Responsible for the Orthoester Linkage Formation in Hygromycin B Biosynthesis as Proved In vivo. Based on previous analysis of the hygromycin B biosynthetic gene cluster (GenBank accession number: AJ628642.1) (Figure 2a), the predicted oxidase HygX is the most promising candidate for catalysis of orthoester formation.18 We therefore aimed to remove the hygX gene from the hygromycin B producing strain Streptomyces hygroscopicus subsp. hygroscopicus DSM 4057823 by in-frame deletion (Figure S1). However, when attempting to approach it through conjugation, tremendous difficulty was encountered for achieving exconjugants and the double crossover mutant. We finally obtained ∆hygX strain after several months of repeated experiments. Analysis by liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (LC-ESI-HRMS) showed

the

presence

of

hygromycin

B

(1)

with

m/z

528.2392

([C20H37N3O13+H]+, calcd. 528.2399) in wild-type (Figure S2a), but it was completely abolished in ∆hygX (Figure 3a). To verify that this was caused by the specific absence of hygX, wild-type hygX was inserted into plasmid 4


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pWHU77 under the control of the PermE* promotor and transferred into ∆hygX to generate ∆hygX::pWHU2848 (Table S1 and S2). However, 1 was still missing in this complementation strain (Figure 3a). Considering that PermE* might not work in DSM 40578, we constructed an alternative version of the complementation plasmid ∆hygX::pWHU2849 (Table S1 and S2) housing a longer fragment containing hygX and a 81 bp sequence upstream of its start codon, which should house the native promotor. In this case, the production of 1 was restored, as expected, to a level similar to wild-type (Figure 3a). Therefore, HygX was confirmed to be involved in the biosynthesis but rigorously not verified to be responsible for catalyzing the cyclization step. AKG/Fe(II)-Dependent Enzyme HygX Converts Uncyclized Hygromycin C to Hygromycin B In vitro and Its Enzymatic Characterization. Instead of hygromycin B, the ∆hygX extract was found to contain a new compound with m/z 530.2553 ([C20H39N3O13+H]+, calcd. 530.2556). It was verified as a ring-open metabolite by tandem mass spectrometry (MS/MS) and NMR (Figure S2b, S4 and Table S3) and is named here hygromycin C (2) (Figure 2b). Compound 2 was also detectable as a minor component in wild-type extracts, which is frequently seen as intermediates and shunt metabolites in other aminoglycoside pathways.24 The structure of this newly-identified compound 2 was consistent with it being an intermediate and the substrate for cyclization. To test this, recombinant HygX (Figure S5) was purified from Escherichia coli and used for enzymatic assays in vitro. As a member of the 5



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AKG/Fe(II)-dependent enzyme family25,26 HygX requires Fe2+ as cofactor.18 Therefore it was pre-incubated with 0.1 mM Fe2+ for 15 min followed by an overnight reaction with 2 and α-ketoglutarate. As shown, 2 was converted to 1 by HygX with a conversion rate of 95% (Figure 3b). This reaction did not occur if

either

Fe2+

or

α-ketoglutarate

was

absent.

Considering

AKG/Fe(II)-dependent enzymes are generally sensitive to oxidative damage18 which may be induced by oxygen, HygX was pre-incubated with Fe2+ in anaerobic condition before exposed to air for catalysis. As expected, the conversion rate in such condition was improved, especially when reaction time was short (Table S4). Kinetic parameters of HygX were determined with purified 2 as substrate. High performance liquid chromatography with an evaporative light scattering detector (HPLC-ELSD) was used to monitor the disappearance of hygromycin C. The data were fitted to the integrated form of the Michaelis-Menten equation using Origin 9.0 software, and the calculated KM for 2 was 0.14±0.02 mM and the kcat was 11.04±0.00 s-1 (Figure 4). The kinetic parameters of HygX are comparable to those of TauD, another member of the AKG/Fe(II)-dependent enzyme family, for its substrate (KM=58±0.6 µM and kcat=12.5±0.5 s-1).27 HygF Links 2-DOS and UDP-galactose to Build the Pseudo Disaccharide Scaffold of Hygromycin B. To investigate earlier steps in the biosynthetic pathway of hygromycin B, we firstly screened wild-type extracts for the presence of disaccharide intermediates. A compound with m/z 6


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339.1762 ([C13H26N2O8+H]+, calcd. 339.1762) was detected with fragment m/z 177.04 by MS/MS (Figure S2c). According to the structure of 2, the pseudo disaccharide

intermediate

is

very

likely

to

contain

the

hyosamine

(3-N-methyl-2-DOS, 4) ring and the D-talose ring, and is named here 3-N-methyl-talamine (7) (Figure 2b). Connection of the destomic aldehyde ring to 7 would form 2. There are only two glycosyltransferase-encoding genes within the hygromycin B biosynthetic gene cluster (Figure 2a): HygF belongs to the GT-A fold superfamily and is likely to need a metal ion for its function,28 while HygD belongs to GT-B fold superfamily and needs no cofactor. These two candidates were cloned and expressed in E. coli as recombinant proteins. Since HygF is annotated as a putative galactosyltransferase, an assay using UDP-galactose (the C-2’-epimer of UDP-talose, 11), 2-DOS (3) and HygF with cofactor Mn2+ was carried out. This incubation produced nearly complete conversion to a new species with m/z 325.1618 ([C12H24N2O8+H]+, calcd. 325.1605) after 10 minutes (Figure 5a). Since no enzyme for isomerization was added, this species is speculated to be galacamine (5) (Figure S2d). Another enzyme may epimerize 5 to talamine, for eventual transformation to hygromycin B. Alternatively, given that HygF showed loose substrate specificity and accepted UDP-glucose with a low efficiency (Figure S6), it is possible that UDP-talose is the substrate for HygF, to generate the D-talose ring of talamine directly. In contrast, HygD showed no catalytic activity in this assay, and is a strong candidate as the enzyme responsible for adding the 7



destomic aldehyde ring. There are two putative epimerases or dehydrogenases, HygJ and HygK, in the hyg cluster that might generate UDP-galactose as the substrate for HygF (Figure 2). In enzymatic assays, HygK was found to interconvert UDP-glucose and UDP-galactose through C-4' epimerization, and the reaction reached an equilibrium favoring UDP-glucose after overnight incubation (Figure S7). This balance may act to prevent depletion of the intracellular UDP-glucose pool. In contrast, HygJ did not catalyze this interconversion. We tested whether HygJ can epimerize 5 to talamine in vitro but no new peak was observed. Since 5 and talamine have the same MS and MS/MS spectra, these two species can hardly be distinguished if they also have similar retention time. Therefore, pending further analysis, HygJ is still a candidate to catalyze epimerization, as the only other predicted epimerase encoded in the cluster. The 3-N Methylation by HygM Occurs Independently with the Formation of Pseudo Disaccharide. Methylation is a universal decoration in natural products that may significantly influence the biological activity and protect the molecule from destructive modification caused by resistance. A unique putative methyltransferase-encoding gene hygM from the hyg cluster (Figure 2a) was cloned, expressed (Figure S5) and tested in vitro. When using 3 and S-adenosyl methionine (SAM) as substrates, a new species with m/z 177.1234 ([C7H16N2O3+H]+, calcd. 177.1234) was detected, indicating a single methylation (Figure 5b). Taking the structures of 2 and 1 as a reference, the 8


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methylation site is most likely to be at N-3 to give 4. To determine the positions of HygM and HygF in the pathway, sequential reactions were performed, in which one enzyme was first added for reaction, followed by heating for enzyme inactivation, and then the other one was supplied for further catalysis. Surprisingly, 3-N-methyl-galacamine (6) (Figure S2e) was obtained in both assays, which showed that 3-N-methylation and disaccharide formation are relatively independent to each other in vitro (Figure 5c and 5d). This result suggested that demethyl homologues of hygromycin B might be formed. Accordingly, 3-N-demethyl-hygromycin C (8) (Figure S2f) in the ∆hygX strain, and 3-N-demethyl-hygromycin B (9) (Figure S2g) in the wild-type strain, were detected on LC-ESI-HRMS and verified by MS/MS. Moreover, 8 was also converted to 9 by HygX in vitro under the same conditions as 2 gave 1 (Figure 3c). Therefore, intermediates lacking N-3 methylation are fully processed to 3-N-demethyl-hygromycin B (9) (Figure 2b), just as gentamicins lacking C-6’ methylation

are

also

fully

processed.24

To

verify

the

function

of

glycosyltransferase hygF and methyltransferase hygM in vivo, we repeatedly attempted to delete them in-frame but we failed to obtain these mutants because of the extreme intractability of the DSM 40578 strain.29 Sedoheptulose-7-phosphate, which is involved in the biosynthesis of lipopolysaccharide,30–32 may serve as the origin of the UDP-destomic acid moiety of hygromycin B. Recently, the biosynthesis of an intermediate, D-glycero-D-altro-heptose-7-phosphate,

was demonstrated by enzymatic

9



assays,

and

its

following

putative

Page 10 of 30

transformation

to

NDP-D-glycero-D-altro-heptose was also predicted.29 For further decoration, epimerization may be introduced and the amino group at C-6’’ site is speculated to be installed through successive dehydrogenation and transamination to form the NDP-destomic aldehyde moiety for linkage to the pseudodisaccharide, accompanied by specific epimerization and optional methylation HygX would then close the orthoester ring to give the final products. In summary, we have characterized the unusual orthoester linkage formation carried out by AKG/Fe(II)-dependent oxygenase HygX in vivo and in vitro, and we have identified the elusive direct precursor of hygromycin B. The independent

formation

of

the

pseudodisaccharide

moiety

and

of

3-N-methylation is also described in vitro, which implies alternative pathways in hygromycin B biosynthesis. These findings expand our knowledge of oxidative cyclization performed by enzymes and of the biosynthesis of aminoglycoside antibiotics, a prerequisite for confident application of synthetic biology to the engineering of these pathways.

10


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METHODS

Bacterial strains, chemicals, and culture conditions. E. coli DH10B was used as cloning host and E. coli ET12567/pUZ8002 for intergeneric conjugation between E. coli and Streptomyces. Streptomyces hygroscopicus subsp. hygroscopicus DSM 40578 wild-type strain was achieved from China General Microbiological Culture Collection Center (CGMCC). Restriction endonucleases, Phusion High-Fidelity Master Mix with GC-buffer, and Gibson Assembly® Master Mix were obtained from New England Biolabs. Oligonucleotide primers were synthesized by GenScript and Tsingke. DNA sequencing of PCR products was performed by GenScript or Tsingke. DIG DNA labeling and detection kits were purchased from Roche. Hygromycin B and 2-deoxystreptamine dihydrobromide were purchased from Sigma-Aldrich, and uridine 5’-diphosphogalactose disodium was from Coolaber. DSM 40578 wild-type and mutants were grown in SFM solid medium (soya flour 3%, D-mannitol 2%, agar 2%) and TSBY liquid medium (tryptone soya broth 3%, yeast extraction 0.5%, sucrose 10.5%) for chromosomal DNA isolation and preparation of mycelium, respectively. E. coli strains were maintained in 2×TY media (yeast extraction 1%, tryptone 1.6%, NaCl 0.5%) at 37°C with appropriate antibiotic selection at a final concentration of 100 µg/mL ampicillin, 25 µg/mL chloramphenicol and 50 µg/mL kanamycin. Construction of hygX disruption plasmids and in-frame deletion mutant. To construct plasmid for in-frame deletion of hygX, two DNA fragments flanking hygX were amplified from the genomic DNA of DSM 40578 by using primers hygX-L1/L2 and hygX-R1/R2 (Table S5). The PCR products were cloned into the Streptomyces-E. coli shuttle vector pYH733 by Gibson 11



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assembly34 to obtain the gene disruption plasmid pWHU2847 (Table S1), which was verified by restriction endonuclease digestion and sequencing. To create in-frame deletion mutant ∆hygX, the corresponding plasmid pWHU2847 was introduced into DSM 40578 by conjugation on a SFM plate (containing 100 mM CaCl2). After incubation at 28°C for 14 h, the plate was overlaid with apramycin

(25

µg/mL)

and

nalidixic

acid

(25

µg/mL).

Single

apramycin-resistant exconjugants from this plate were patched on SFM plate containing apramycin (25 µg/mL) and nalidixic acid (25 µg/mL), and grown at 28 °C for 2 or 3 days. To screen the double cross-over mutant, each single colony from the resistance double check plate was patched onto SFM plates without and with 25 µg/mL apramycin, respectively. Genomic DNA of single apramycin-sensitive colonies was extracted and checked by PCR using the checking primers hygX-PC1 and hygX-PC2 (Table S5), and further confirmed by sequencing and Southern blot analysis (Figure S1). Gene complementation of ∆hygX mutant. For Complementation, pWHU2848 and pWHU2849 (Table S1) were constructed by inserting hygX into vector pWHU7735 under the control of the constitutive promotor PermE* and the native promotor, respectively, by Gibson assembly.34 After sequencing confirmation, the plasmid was introduced into ∆hygX by conjugation. Complemented exconjugant was verified on SFM medium containing thiostrepton (25 µg/mL) and confirmed by PCR with checking primers hygX-CPC1 and hygX-CPC2 (Table S5). Production, extraction and analysis of hygromycin B and related intermediates. For fermentation and detection of hygromycin B and related intermediates, DSM 40578 and mutant were fermented in two stages. A seed 12


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culture was maintained in TSBY liquid medium at 28°C with shaking at 220 rpm for 36 h before being inoculated into liquid ISP2 medium (yeast extraction 0.4%, malt extract 1%, glucose 0.4%, 1% inoculum), then incubated at 28°C with shaking at 220 rpm for 5 days. The fermentation broths were adjusted to pH 2.0 with H2SO4 then agitated for 1 h. The clarified supernatant after centrifugation was filtered through Whatman filter paper, and agitated with DOWEX 50 WX8-200 ion-exchange resin (1 g for 40 mL broth) that was preconditioned with acetonitrile followed by Milli-Q water for 3 times. After 1 h， the resin was put in a column then washed by Milli-Q water (6 column volume) and eluted with 1 M NH4OH (6 column volume). The eluate was freeze-dried and re-dissolved in Milli-Q water (0.2 mL concentrated solution was equivalent to 40 mL broth), and filtered through 0.22 µm microporous membrane before subjection to LC-ESI-HRMS analysis. LC-ESI-HRMS analysis of extracts was performed on a Thermo Electron LTQ-Orbitrap XL fitted with a Phenomenex Luna C18 column (250×4.6 mm) at a flow rate of 0.4 mL/min using a mobile phase of (A) 0.2% trifluoroacetic acid (TFA) in H2O (adjusted to pH 2.0 with NH4OH) and (B) 100% CH3CN. The gradient for separation of hygromycin B and intermediates: 0-2 min 2% B, 2-13 min 2% B to 6% B, 13-13.5 min 6% B-90% B, 13.5-18 min 90% B, 18-18.5 min 90% B to 2% B, 18.5-24 min 2% B. MS/MS analysis were carried out in the positive ionization mode with 35% relative collision energy. Isolation and purification of hygromycin C. Isolation of hygromycin C from crude extract was performed on a Thermo Scientific HPLC (UltiMate 3000) fitted with ELSD (Alltech 2000ES), and a Phenomenex Synergi C18 column (250×10 mm) at a flow rate of 3 mL/min using a mobile phase of (A) 0.2% 13



Page 14 of 30

trifluoroacetic acid in H2O and (B) 100% CH3CN. The gradient for separation: 0-6 min 2% B to 5% B, 6-7 min 5% B to 90% B, 7-11 min 90% B, 11-12 min 90% B to 2% B, 12-16 min 2% B. The temperature and gas flow of ELSD was set 109°C and 2.9 L/min, respectively. NMR characterization of hygromycin C. For structure elucidation of hygromycin C, 1D (1H,

13

HSQC-TOCSY

NOESY)

and

C and DEPT) and 2D (1H-1H COSY, HSQC, HMBC, NMR

spectra were

collected

on

an

Agilent-NMR-vnmrs 600 spectrometer. Chemical shifts were reported in ppm using Tetramethylsilane as an internal reference, and NMR data processing was performed by using MestReNova software. Construction of protein expression plasmids, overexpression and purification of proteins. The target genes hygX, hygM, hygJ, hygK, hygD and hygF were amplified from the genomic DNA of DSM 40578 by PCR using corresponding primers (Table S5) with 30 cycles of denaturation at 98°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds per kilobase plus final extension at 72°C for 10 min. The PCR product was purified by gel extraction, and inserted into vector pET28a(+) between NdeI and EcoRI to create protein expression plasmids pWHU2856, pWHU2857, pWHU2858, pWHU2859, pWHU2860 and pWHU2861 (Table S1). The resulting constructs were verified by restriction endonuclease digestion and DNA sequencing, then used to transform E. coli BL21(DE3). The transformant was grown at 37°C in LB medium (yeast extraction 0.5%, tryptone 1%, NaCl 1%, in addition with 50 µg/mL kanamycin) with shaking until OD600 to 0.4-0.6. After addition of 0.1 mM IPTG for induction, the cultures were continued to shake at 18°C for 16 h, then harvested by centrifugation at 5000 14


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rpm for 15 min, and re-suspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.8). Cells were disrupted by sonication (30 min with a 25% duty cycle), and the lysate was clarified by centrifugation (12000 rpm, 4 °C, 1 h). The supernatant was passed through a 5 mL IMAC column (GE Healthcare) charged with nickel and previously equilibrated with lysis buffer. Proteins were eluted using a linear gradient of imidazole (up to 800 mM) in a buffer of 50 mM Tris-HCl (pH 7.8) and 150 mM NaCl. Fractions containing target proteins were concentrated using Amicon-Ultra Centrifugal Filters (Millipore) and further purified using PD-10 column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 7.8) and 150 mM NaCl. Fractions containing HygX, HygM, HygJ, HygK, HygD and HygF proteins were concentrated to 32 mg/mL, 2 mg/mL, 3 mg/mL, 1 mg/mL, 17 mg/mL and 19 mg/mL, respectively, and stored in 10% glycerol at -80 °C until being used. All these proteins were confirmed by SDS-PAGE. Identification of proteins. The exact mass of proteins HygX, HygM, HygJ, HygK, HygD and HygF without methionine were verified by mass spectrum on a Thermo Electron LTQ-Orbitrap XL fitted with a Phenomenex Jupiter C4 column (250×2 mm) at a flow rate of 0.3 mL/min using a mobile phase of (A) 0.1% TFA in H2O and (B) 0.1% TFA in CH3CN. The gradient for separation of protein: 0-1 min 5% B, 1-20 min 5% B to 95% B, 20-25 min 95% B, 25-27 min 95% B to 5% B, 27-30 min 5% B. The mass spectrometer was set to full scan (from 300 to 2000 m/z). The mass spectrometric data were processed and deconvoluted using the Bioworks software (Thermo Finnigan). Enzymatic assay and kinetic characterization of HygX. The in vitro enzymatic assay of HygX in open air condition or in anaerobic chamber (Coy Labs) was carried out by combining 25 mM Tris-HCl (pH 7.4), 75 mM NaCl, 0.1 15



Page 16 of 30

mM Fe2+ with 10 µM purified recombinant HygX for pre-incubation for 15 min, followed by adding 0.5 mM α-ketoglutarate and 0.15 mM hygromycin C in 200 µL total reaction volume. For the assay to convert 3-N-demethyl-hygromycin C (8) to 3-N-demethyl-hygromycin B (9), the fermentation broth of ∆hygX was applied as substrate. Reaction was typically incubated at 28°C for 3 h followed by treatment with equal volume of chloroform to precipitate protein, and filtered through 0.22 µm microporous membrane for LC-ESI-HRMS analysis. The boiled inactivation protein was used as a negative control. The activity of HygX was determined by following the hygromycin C consumption using HPLC-ELSD. The reaction mixture (1000 µL) for the assay of HygX activity contained enzyme (2.5 µM), α-ketoglutarate (2 mM) and various concentrations of hygromycin C (0.075, 0.150, 0.375, 0.6, 1.3 mM) in 25 mM Tris-HCl buffer (pH 7.4) with 75 mM NaCl. The reaction mixture was incubated at 28 °C for 10 min. Samples (100 µL) were withdrawn every 2 min, and extracted with equal volumes of chloroform which was filtered through 0.22 µm

microporous

membrane

before

HPLC-ELSD

analysis.

Each

experiment was repeated three times. Enzymatic assay of HygM. The in vitro enzymatic assay of HygM was carried out by combining 25 mM Tris-HCl (pH 7.4), 75 mM NaCl, 0.16 mM S-adenosyl methionine (SAM), 0.1 mM 2-DOS (3) and 3.5 µM purified recombinant HygM in 200 µL total reaction volume. The reaction mixture was incubated at 28°C for 3 h followed by treatment with equal volume of chloroform to precipitate protein, then filtered through 0.22 µm microporous membrane before subjection to LC-ESI-HRMS analysis. The boiled inactivation HygM was used as a negative control. To determine the reaction 16


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order of HygM and HygF, the above HygM assay was abolished in boiling water then 0.6 µM HygF was added with 50 µM Mn2+ and 0.1 mM UDP-galactose for the further incubation at 28°C for 3 h and LC-ESI-HRMS analysis. Enzymatic assay of HygF. The in vitro enzymatic assay of HygF was carried out by combining 25 mM Tris-HCl (pH 7.4), 75 mM NaCl, 50 µM MnCl2, 0.1 mM 2-DOS, 0.1 mM UDP-galactose and 0.6 µM purified recombinant HygF in 200 µL total reaction volume. The reaction mixture was incubated at 28°C for 3 h followed by treatment with equal volume of chloroform to precipitate protein, and filtered through 0.22 µm microporous membrane before subjection to LC-ESI-HRMS analysis. The boiled inactivation protein was used as a negative control. To determine the reaction order of HygF and HygM, the above HygF assay was abolished in boiling water then 3.5 µM HygM was added with 0.16 mM SAM for the further incubation at 28°C for 3 h and LC-ESI-HRMS analysis. Enzymatic assays of HygJ and HygK. The in vitro enzymatic assay of HygJ or HygK was carried out by combining 25 mM Tris-HCl (pH 7.4), 75 mM NaCl, 0.4 mM NAD+/NADP+, 0.2 mM UDP-glucose/UDP-galactose and 0.6 µM purified recombinant HygJ/HygK in 50 µL total reaction volume. The reaction mixture was incubated at 28°C for overnight followed by treatment with equal volume of chloroform. The boiled inactivation protein was used as a negative control. The analysis of UDP-hexose as substrates or products was performed on HPLC connected to a SPD-M20A ultraviolet absorption detector (Shimadzu) fitted with a COSMOSIL PBr column (250×4.6 mm) at a flow rate of 0.8 mL/min. The mobile phase was sodium phosphate solution (100 mM, pH 7.0). 17



UDP-hexose showed a significant absorption at wavelength of 254 nm.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXX. Table S1-S5 and Figure S1-S7

AUTHOR INFORMATION Correspondence Author *E-mail: [email protected] ORCID Yuhui Sun: 0000-0002-9258-2639 Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China (31470186). We thank Z. Ding at Yunnan University for his help in NMR analysis of hygromycin C and P. F. Leadlay at University of Cambridge for his critical reading of the manuscript.

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REFERENCES (1)

Mann, R. L., and Bromer, W. W. (1958) The isolation of a second antibiotic from Streptomyces hygroscopicus. J. Am. Chem. Soc. 80, 2714–2716.

(2)

Cameron, A., and Gaynor, E. C. (2014) Hygromycin B and apramycin antibiotic resistance cassettes for use in Campylobacter jejuni. PLoS ONE 9:e95084.

(3)

Radman, I., Greiss, S., and Chin, J. W. (2013) Efficient and rapid C. elegans transgenesis by bombardment and hygromycin B selection. PLoS ONE 8:e76019.

(4)

Blochlinger,

K.,

phosphotransferase

and as

Diggelmann, a

selectable

H.

(1984)

marker

for

Hygromycin DNA

B

transfer

experiments with higher eucaryotic cells. Mol. Cell. Biol. 4, 2929–2931. (5)

Macintyre, G., Woods, D. E., and Anderson, R. (1991) Hygromycin B inhibits synthesis of murine coronavirus RNA. Antimicrob. Agents Chemother. 35, 2630–2633.

(6)

Pfister, P., Risch, M., Brodersen, D. E., and Böttger, E. C. (2003) Role of 16S rRNA helix 44 in ribosomal resistance to hygromycin B. Antimicrob. Agents Chemother. 47, 1496–1502.

(7)

McGaha, S. M., and Champney, W. S. (2007) Hygromycin B inhibition of protein synthesis and ribosome biogenesis in Escherichia coli. Antimicrob. Agents Chemother., 51, 591–596. 19



(8)

Page 20 of 30

Eustice, D. C., and Wilhelm, J. M. (1984) Mechanisms of action of aminoglycoside antibiotics in eucaryotic protein synthesis. Antimicrob. Agents Chemother. 26, 53–60.

(9)

Carter, A. P., Clemons, W. M., Brodersen, D. E., Morgan-Warren, R. J., Wimberly, B. T., and Ramakrishnan, V. (2000) Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 407, 340–348.

(10) Borovinskaya, M. A., Shoji, S., Fredrick, K., and Cate, J. H. D. (2008) Structural basis for hygromycin b inhibition of protein biosynthesis. RNA 14, 1590–1599. (11) Kudo, F., Yamamoto, Y., Yokoyama, K., Eguchi, T., and Kakinuma, K. (2005) Biosynthesis of 2-deoxystreptamine by three crucial enzymes in Sreptomyces fradiae NBRC 12773. J. Antibiot. 58, 766–774. (12) Weitnauer, G., Mühlenweg, A., Trefzer, A., Hoffmeister, D., Süßmuth, R. D., Jung, G., Welzel, K., Vente, A., Girreser, U., and Bechthold, A. (2001) Biosynthesis of the orthosomycin antibiotic avilamycin A: deductions from the molecular analysis of the avi biosynthetic gene cluster of Streptomyces

viridochromogenes

Tü57

and

production

of

new

antibiotics. Chem. Biol. 8 , 569–581. (13) Weitnauer, G., Gaisser, S., Kellenberger, L., Leadlay, P. F., and Bechthold, A. (2002) Analysis of a C-methyltransferase gene (aviG1) involved

in

avilamycin

biosynthasesis 20


in

Streptomyces

Page 21 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


viridochromogenes Tü57 and complementation of a Saccharopolyspora erythraea eryBIII mutant by aviG1. Microbiology 148, 373–379. (14) Hofmann, C., Boll, R., Heitmann, B., Hauser, G., Dürr, C., Frerich, A., Weitnauer, G., Glaser, S. J., and Bechthold, A. (2005) Genes encoding enzymes responsible for biosynthesis of L-Lyxose and attachment of eurekanate during avilamycin biosynthesis. Chem. Biol. 12, 1137–1143. (15) Vey, J. L., Al-Mestarihi, A., Hu, Y., Funk, M. A., Bachmann, B. O., and Iverson, T. M. (2010) Structure and mechanism of orf36, an amino sugar oxidizing enzyme in everninomicin biosynthesis. Biochemistry 49, 9306– 9317. (16) Arenz, S., Juette, M. F., Graf, M., Nguyen, F., Huter, P., Polikanov, Y. S., Blanchard, S. C., and Wilson, D. N. (2016) Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome. Proc. Natl. Acad. Sci. U. S. A. 113, 7527–7532. (17) Tang, M. C., Zou, Y., Watanabe, K., Walsh, C. T., and Tang, Y. (2017) Oxidative cyclization in natural product biosynthesis. Chem. Rev. 117, 5226–5333. (18) McCulloch, K. M., McCranie, E. K., Smith, J. A., Sarwar, M., Mathieu, J. L., Gitschlag, B. L., Du, Y., Bachmann, B. O., and Iverson, T. M. (2015) Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily. Proc. Natl. Acad. Sci. U. S. A. 112, 11547–11552. 21



Page 22 of 30

(19) Wang, C., Chang, W. C., Guo, Y., Huang, H., Peck, S. C., Pandelia, M. E., Lin, G. M., Liu, H. W., Krebs, C., and Bollinger, J. M. (2013) Evidence that the fosfomycin-producing epoxidase, HppE, is a non-heme-iron peroxidase. Science 342, 991–995. (20) Strieker, M., Kopp, F., Mahlert, C., Essen, L. O., and Marahiel, M. A. (2007)

Mechanistic

and

structural

basis

of

stereospecific

cβ-hydroxylation in calcium-dependent antibiotic, a daptomycin-type lipopeptide. ACS Chem. Biol. 2, 187–196. (21) Blasiak, L. C., Vaillancourt, F. H., Walsh, C. T., and Drennan, C. L. (2006) Crystal structure of the non-haem iron halogenase SyrB2 in syringomycin biosynthesis. Nature 440, 368–371. (22) Zhang, Z., Ren, J., Stammers, D. K., Baldwin, J. E., Marios, K., and Schofield, C. J. (2000) Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase. Nat. Struct. Biol. 7, 127–133. (23) Komaki, H., Ichikawa, N., Oguchi, A., Hamada, M., Tamura, T., Fujita, N., and Suzuki, K. I. (2017) Genome analysis-based reclassification of Streptomyces heterotypic

endus synonyms

and of

Streptomyces Streptomyces

sporocinereus hygroscopicus

as

later subsp.

hygroscopicus. Int. J. Syst. Evol. Microbiol. 67, 343–345. (24) Guo, J., Huang, F., Huang, C., Duan, X., Jian, X., Leeper, F., Deng, Z., Leadlay, P. F., and Sun, Y. (2014) Specificity and promiscuity at the 22


Page 23 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


branch point in gentamicin biosynthesis. Chem. Biol. 21, 608–618. (25) Kovaleva, E. G., and Lipscomb, J. D. (2008) Versatility of biological non-heme Fe(II) centers in oxygen activation reactions. Nat. Chem. Biol. 4, 186–193. (26) Wu, L. F., Meng, S., and Tang, G. L. (2016) Ferrous iron and α-ketoglutarate-dependent dioxygenases in the biosynthesis of microbial natural products. Biochim. Biophys. Acta. 1864, 453–470. (27) Grzyska, P. K., Ryle, M. J., Monterosso, Liu, G. R., J., Ballou, D. P., and Hausinger, R. P. (2005) Steady-state and transient kinetic analyses of taurine/alpha-ketoglutarate

dioxygenase:

effects

of

oxygen

concentration, alternative sulfonates, and active-site variants on the FeIV-oxo intermediate. Biochemistry 44, 3845–3855. (28) Hu, Y., and Walker, S. (2002). Remarkable structural similarities between diverse glycosyltransferases. Chem. Biol. 9, 1287–1296. (29) Tang, W., Guo, Z., Cao, Z., Wang, M., Li, P., Meng, X., Zhao, X., Xie, Z., Wang,

W.,

and

Zhou,

A.

D-sedoheptulose-7-phosphate

Lou,

C.,

and

Chen,

Y.

(2018)

is a common precursor for the heptoses

of septacidin and hygromycin B. Proc. Natl. Acad. Sci. U. S. A. 115, 2818–2823. (30) Taylor, P. L., Blakely, K. M., De Leon, G. P., Walker, J. R., McArthur, F., Evdokimova, E., Zhang, K., Valvano, M. A., Wright, G. D., and Junop, M. S. (2008) Structure and function of sedoheptulose-7-phosphate 23



Page 24 of 30

isomerase, a critical enzyme for lipopolysaccharide biosynthesis and a target for antibiotic adjuvants. J. Biol. Chem. 283, 2835–2845. (31) Valvano, M. A., Messner, P., and Kosma, P. (2002) Novel pathways for biosynthesis of nucleotide-activated glycero-manno-heptose precursors of bacterial glycoproteins and cell surface polysaccharides. Microbiology 148, 1979–1989. (32) Yu, C. K., Wang, C. J., Chew, Y., Wang, P. C., Yin, H. S., and Kao, M. C. (2016) Functional

characterization of

helicobacter

pylori

26695

sedoheptulose 7-phosphate isomerase encoded by hp0857 and 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., 24


Page 25 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.

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Figure 1. The representative natural products containing orthoester linkages. The orthoester linkages in each compound are highlighted in red.


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Figure 2. Biosynthetic gene cluster and proposed model for hygromycin B biosynthesis. (a) The organization of hygromycin B biosynthetic gene cluster from DSM 40578. (b) The 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 in blue, red and green are building blocks related to hyosamine, D-talose and destomic acid rings of hygromycin B biosynthesis, respectively.



Figure 3. The genetic and enzymatic analysis of orthoester moiety formation catalysed 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 4. Kinetic constants of AKG/Fe(II)-dependent oxidase HygX. (a) Standard curve of hygromycin C, here lgC and lgA represent the natural logarithm of hygromycin C concentration (25-2000 µM) and peak area, respectively. (b) Michaelis–Menten saturation curve of HygX. Error bars represent standard deviation of means.


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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 followed by HygM, and (e) no enzyme as control. Traces in blue, green yellow and red indicate the extracted ion chromatograms of 2-DOS (3), hyosamine (4), galacamine (5) and 3-N-methylgalacamine (6), respectively, in each panel.


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Direct Genetic and Enzymatic Evidence for Oxidative Cyclization in

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