Materials and Methods

2.1 Gene cloning and protein purification

The dbv29 gene was amplified from genomic DNA by PCR. The products were each ligated according to the manufacturers' instruction (Novagene) into the pET-28a(+) expression vector which provides an N-terminal His6-tagged protein. E. coli cells were grown at 37°C in 1 L of LB medium until an A₆₀₀ of about 0.7 was reached. Protein expression was induced with 0.2 mM IPTG at 16°C, and, after 12 h, the cells were harvested by centrifugation, resuspended in 10 mM imidazole-HCl buffer, pH 7.8 (binding buffer), and ruptured using a microfluidizer. The cell-free extract prepared by centrifugation was applied to an Ni²⁺- resin column, which was then washed successively with binding buffer and wash buffer before eluting the target protein with m imida ole- Cl. Gel filtration was performed using an kta FPLC system equipped with an S-200 Superdex column (Amersham Bioscience) under isocratic conditions (20 mM Tris, pH 7.6, 100 mM KCl). The buffer was exchanged for 50 mM HEPES buffer, pH 7.2 using Millipore centrifugal filters. Protein concentrations were estimated using the Bradford assay. The purified protein was confirmed by SDS-PAGE, Western blotting, and electrospray mass spectrometry (ESI-MS).

2.2 Crystallization and data collection

Dbv29 was crystallized by using hanging drop vapor diffusion method at 20 °C. 10 mg ml⁻¹ Dbv29 in 20 mM TRIS (pH 8.0, 100 mM NaCl) was mixed with the same volume of reservoir solution. Hexagonal crystals can be obtained in two crystallization conditions. One contains 50 mM calcium chloride, 100 mM BIS-TRIS (pH 6.5), 30%

(v/v) PEGMME 550; the other contains 200 mM imidazole malate (pH 5.5), 33% (v/v) PEG 600. Crystals were obtained after one-week incubation. A different crystal shape was achieved in 200 mM di-ammonium hydrogen citrate (pH 5.0), 17% (w/v) PEG

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3350, C10 teicoplanin (5 mM; C10 teicoplanin was purified from commercially available teicoplanin mixtures by HPLC; the purified C10 teicoplanin was salt-free, so it required 10% (v/v) DMSO to dissolve), whereby a crystal was obtained after a five-month incubation. Data sets were collected on an ADSC Quantum-210 CCD using the synchrotron radiation -ray source tuned at a wavelength of . and an operating temperature of 100 K at beamline 13C1 of the National Synchrotron Radiation Research Center in Taiwan. Data were indexed and scaled with the HKL2000 package²⁰. The hexagonal crystal belongs to P6₁22 space group with unit cell dimensions a b 66. and c . . ecause of the large cell dimensions, the maximum resolution that can be reached is . . The crystal obtained from the second condition belongs to P21 space group with unit cell dimensions a = 61. , b 0.8 , c = 124.9 and β 8.4° and was diffracted to 1.93 resolution. he contents of both asymmetric units were estimated from the Matthews coefficient²¹. The data suggest that a value of . ³ Da^-1 with 40.8% solvent corresponds to two molecules per asymmetric unit in the P6122 crystal, and a value of 2.44 ³ Da⁻¹ with 49.7% solvent content indicates four molecules per asymmetric unit in the P2₁ crystal.

2.3 Structure determination and refinement

The molecular replacement method was used to obtain phase information, and MolRep²² was used to find the phase solution. nitial phases were obtained at resolution using the coordinates of AknOx (PDB ID: 2IPI) as the search model. Phase extension yielded electron density maps into which a polypeptide model was built with the program XtalView²³. The model was further refined with CNS and REFMAC^{24, 25}. Water molecules were added with a water-pick routine in the CNS program. The final model has an factor of 6. for all reflections between and . resolution and an R_free of 20.5% under 5% randomly distributed reflections. In the Ramachandran

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plot, 99.5% of all residues are in the allowed region. Figures were generated using PyMOL (http://www.pymol.org). Detailed refinement statistics are given in Table 2.

2.4 Enzymatic activity assay

Dbv29 activity was determined by LC-MS. The assay mix containing en yme ( μg) and the corresponding substrate (1 mM) in buffer (50 mM HEPES pH 7.2, 100 mM aCl, m D ) (total volume μl) was incubated for 2 h at 37 °C. Each reaction mixture was then centrifuged at 16,000 g for 5 min (Heraeus Biofuge Pico) and filtered on an ultracentrifugal filter unit (5 kDa cut-off membrane, Millipore). The filtrate was directly subjected to HPLC-ESI/Q-Tof (Waters HPLC 2695 interfaced with an ESI source coupled to a Micromass micro Q-Tof mass spectrometer) or HPLC-ESI-LTQ (Agilent 1200 Series interfaced with an ESI source coupled to a Thermo-Finnigan LTQ XL ion trap spectrometer), using a gradient of 0–60% acetonitrile in 0.1% TFA in water over 30 min. Online LC-MS spectra were recorded by MassLynx (Waters) or Xcalibur (Thermo Fisher Scientific, Inc.).

2.5 Mutagenesis

Site-directed mutagenesis was carried out by using QuickChange (Stratagene), and the wild-type Dbv29 was used as the template for single mutation. For double mutation, the single or double mutants, respectively, served as templates. All mutations were confirmed by DNA sequencing. The pET/His plasmid was used for protein expression.

Mutant enzymes were purified with the same protocol as recombinant wild-type Dbv29.

2.6 Circular dichroism spectroscopy

For structural analysis, far UV circular dichroism spectra of wild-type Dbv29 and mutants were recorded between 240 nm and 190 nm on a Jasco circular dichroism spectropolarimeter at 25 °C. Proteins were present at 1 mg ml⁻¹ in a standard buffer

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solution. Each spectrum represents an average of three scans, wherein the buffer background is subtracted. For stability analysis, proteins of wild-type Dbv29 and Y165F/Y473F mutant were dissolved in a standard buffer solution at 1 mg ml⁻¹ for thermal melting experiments. Circular dichroism measurement was carried out with a Jasco J-815 spectropolarimeter in a temperature range of 20–100 °C at 222 nm wavelength. Melting temperatures, Tm, were determined by using two-state approximation.

2.7 Analytical ultracentrifuge analysis

The sedimentation velocity experiments were performed with a Beckman-Coulter XL-I analytical ultracentrifuge. Samples and buffers were loaded into 12-mm standard double-sector Epon charcoal-filled centerpieces and mounted in an An-60 Ti rotor. We introduced μl of a mg ml⁻¹ sample into the cell. Sedimentation velocity experiments were performed at rotor speed of 40,000 r.p.m. at 20 °C. The signals of samples were monitored at 280 nm and collected every 3 min for 6 h. The raw data of experiments were calculated using SedFit software²⁶. The density and viscosity of buffer were calculated using Sednterp software (http://

www.jphilo.mailway.com/default.htm). All samples were visually checked for clarity after ultracentrifugation, and no indication of precipitation was observed.

2.8 Synthetic conditions for new analogs

Teicoplanin (a mixture of five analogs) was purchased from AAPIN Chemicals Ltd (UK). The mixture was subjected to HPLC purification to obtain C10-Teicoplanin with purity >95%. 5-Azide pentylamine (99%) was obtained from Dr. Chung-Yi Wu (Academia Sinica) as a gift. All other chemicals were obtained from Sigma/Aldrich Chemical Co. (St. Louis, MO, USA) without further purification unless otherwise stated.

Teicoplanin analogs (2-6) were enzymatically synthesized as described previously9, 10, 14,

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15. In brief, compound 2 was prepared by adding Dbv21 or Orf2* (10 g) into a typical buffer solution (50 mM HEPES, pH 7.2, 100 mM NaCl, 1 mM DTT) containing Tei (1, 1 mM) for 5 h at 37°C; compounds 3-6 were prepared by adding Dbv8 (10 g) into the same buffer solution but containing compound 2 and butanoyl-, hexanoyl-, octanoyl-, or decanoyl-CoA for compounds 3-6, respectively, at 25°C overnight. For the final reaction, Teicoplanin analogs (9, 10, 11, 25 and 32, which showed relatively higher yields from initial tests) were enzymatically synthesized using the optimized conditions described below. Enzyme stability (and, indirectly, the extent of exposure of the aldehyde for functionalization) was tested in an array of organic solvents (MeCN, MeOH, EtOH, DHF, DMF, DCM, DMSO etc.); DMSO turned out to be the most appropriate solvent as Dbv29 is highly stable in up to 90% DMSO. 50% DMSO, however, is included in the current protocol as it gives relatively higher yields. Other conditions, such as the amounts of alkylamine and reductant²⁷, were also optimized one at a time against the fixed concentration of Tei (0.5 mM) for a better yield (below). In general, 10-fold of alkylamine and the reducing agent versus Tei was determined to be most appropriate for the current protocol, which is summarized as follows: Tei (0.5 mM), alkylamine (5 mM; carbon number >6), and Na(CN)BH₃ (5 mM) in 50% DMSO buffer solution at 37°C overnight incubation.

2.9 Compound characterization

All compounds were characterized by MS and HPLC. For select compounds, NMR analyses were performed on a Bruker Avance 600 spectrometer equipped with CryoProbe^TM, with tetramethylsilane (TMS) as an internal standard. Compounds were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) if not otherwise stated, and spectra were recorded at room temperature. NMR peaks of reference compound

(de-r4-11

Tei) and compound 25 are listed below (also see Table 5); original spectra can be found in Figures 11 and 12.

Reference compound (de-r4-Tei). HRMS ES(-): 780.1677 [M-2H]^-2, calc. for C72H68O28N8Cl2 780.1760 [M-2H]^-2 (Figure 13). ¹H-NMR (600 MHz, DMSO-d6) (Figure 11): 9.68 (m, 2H), 9.27 (m, 1H), 8.65 (m, 1H), 8.53 (s, 1H), 8.42 (s, 1H), 7.92 (m, 2H), 7.80 (m, 1H), 7.26 (m, 3H), 7.18 (m, 2H), 7.10 (m, 4H), 6.97 (m, 1H), 6.75 (m, 3H), 6.46 (m, 2H), 6.23 (m, 1H), 5.27 (m, 2H), 5.13 (m, 2H), 4.42 (m, 2H), 4.34 (m, 1H), 4.28 (d, J 11.5, 1H), 4.12 (m, 1H), 3.06 (m, 2H), 2.95 (m, 1H), 1.88 (m, 2H), 1.26 (m, 1H). ¹³C NMR (600 MHz, DMSO-d6): 172.79, 172.72, 169.87, 169.63, 169.32, 168.59, 168.44, 167.36, 167.27, 167.19, 158.76, 158.67, 158.56, 158.49, 158.29, 158.08, 157.87, 157.46, 157.38, 155.52, 155.09, 150.27, 150.23, 149.06, 148.56, 148.49, 147.63, 143.51, 135.58, 134.67, 134.41, 128.26, 128.19, 127.13, 126.20, 125.66, 125.25, 123.43, 120.71, 120.19, 120.08, 118.29, 116.30, 105.66, 99.26, 99.13, 97.52, 96.65, 76.91, 76.81, 73.80, 73.66, 73.00, 72.55, 70.59, 70.00, 69.92, 69.81, 65.99, 65.92, 63.11, 61.13, 60.75, 59.25, 56.12, 56.07, 55.86, 55.79, 55.17, 54.59, 23.21, 23.12. An expanded version of HSQC, HMBC and ¹H COSY spectra of the reference compound is shown in Figure 11.

Compound 25. HRMS ES(+): 984.3033 [M+2H]²⁺, calc. for C₉₅H₁₀₄O₃₂N₁₀Cl₂ 984.3098 [M+2H]²⁺ (Figure 13). ¹H-NMR (600 MHz, DMSO-d6) (Figure 12): 9.67 (m, 2H), 8.61 (m, 1H), 8.40 (m, 1H), 7.93 (m, 2H), 7.77 (m, 2H), 7.18 (m, 14H), 6.71 (m, 3H), 6.48 (m, 2H), 6.23 (m, 1H), 5.24 (m, 7H), 4.96 (m, 1H), 4.62 (m, 1H), 4.39 (m, 5H), 4.08 (m, 1H), 3.70 (m, 2H), 3.00 (m, 4H), 2.14 (m, 1H), 1.99 (m, 1H), 1.86 (m, 3H), 1.48 (m, 2H), 1.37 (m, 1H), 1.15 (m, 12H), 0.84 (d, J 6.5, 6H). ¹³C NMR (600 MHz, DMSO-d₆):

172.87, 172.79, 172.41, 172.36, 169.94, 169.81, 169.39, 169.24, 168.88, 168.76, 168.35, 167.28, 158.54, 158.41, 158.21, 158.00, 157.80, 157.67, 157.48, 157.41, 155.59, 148.27,

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135.73, 135.62, 134.68, 131.88, 131.82, 130.21, 130.09, 129.96, 129.88, 129.20, 129.12, 128.86, 128.80, 128.38, 125.02, 120.79, 120.74, 120.20, 118.28, 116.29, 114.62, 109.20, 98.91, 96.62, 76.95, 76.83, 73.80, 73.73, 72.85, 72.80, 72.71, 72.56, 72.44, 72.20, 70.76, 70.68, 70.09, 69.81, 66.02, 63.13, 61.30, 61.20, 61.08, 60.76, 56.58, 56.38, 56.19, 55.77, 54.57, 51.74, 51.00, 50.16, 35.98, 31.37, 29.23, 29.10, 29.02, 28.97, 28.92, 28.87, 28.80, 27.47, 27.40, 26.76, 26.71, 26.67, 25.11, 24.97, 23.25, 23.17, 22.65, 22.18,14.09. An expanded version of HSQC, HMBC and ¹H COSY spectra of the compound 25 is shown in Figure 12.

2.10 In vivo study

ICR female mice were purchased from the National Laboratory Animal Breeding and Research Center, Taipei, Taiwan. Mice with average body weight of 27 to 30 g were subjected to infection via intravenous (i.v.) injection with 1.3 × 10⁵ cfu/mouse of E.

faecalis (ATCC 51559) at day 0. For treatment study, mice were randomized into four groups at the start of the experiment and administered 10 mg/kg of either vancomycin, teicoplanin, benzylamine-teicoplanin (25) or saline (control) by i.v. twice a day for three days (from day 1 to day 3, a total of 6 doses). Mice were subjected to anesthesia; whole blood was then sampled from orbital sinus on day 1, day 2, and day 3. The whole blood underwent serial dilutions with PBS, which were plated on Brain Heart infusion agar (BHI agar; Difico, Detroit, MI, USA) for enumeration of cfu (colony formation unit).

The graphs and statistical analyses were performed using SigmaPlot® and SigmaStat® . Differences were considered significant if the P value was < 0.05.

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