Supporting Information

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SI Materials and Methods

The ORF encoding the MiaE protein was PCR amplified using S. typhimurium genomic DNA, Pwo polymerase (Roche) and the following primers: the N terminus primer, which contained the first AUG start codon, located 11 bp downstream of the stop codon of the preceding ORF (miaE-N1: 5'-AGCTGCATCATATGACGGTAC-3') was designed to contain a unique NdeI restriction site at the predicted initiation codon and the C terminus primer (miaE-C: 5'-AAACGCTAAGCTTTAATTTTACCC-3') complementary to part of the 3'-untranslated region and containing a unique HindIII restriction site. For the second possible AUG start codon located 49 bp downstream of the same stop codon the following primer was used (miaE-N2: 5'-CAGGTTACATATGAATTACCCGCTTTATG-3'). The bold and italicized nucleotide CATATG and AAGCTT concern the unique NdeI and HindIII restriction sites, respectively. The two PCR fragments were purified using the High Pure PCR kit (Roche), double digested with NdeI and HindIII (Fermentas) and gel-purified before direct cloning into the pT₇-7 expression vector, generating to the pT₇-miaE1 and pT₇-miaE2 plasmids. The N-terminal hexahistidine-tagged MiaE proteins were obtained as described (1). The pT₇-miaE1 and pT₇-miaE2 derived plasmids containing the hexahistidine sequence were named pT₇-miaE1H and pT₇-miaE2H. DH5a cells were transformed with the two plasmids and one clone of each, containing an insert with the expected size, was selected for sequencing.

To produce MiaE2H in sufficiently soluble form, an overnight culture of BL21(DE3) cells previously transformed with pT₇-miaE2H was used to inoculate fresh M9 medium containing 0.5 mM citrate-iron, 2 mM MgSO₄, 0.1 mM CaCl₂, 0.4% glucose, 2 mg/ml thiamine and 2% casamino-acids. The cells were grown at 37°C with 100 mg/ml ampicillin. When OD₆₀₀ reached 0.7, expression of the recombinant protein MiaE2H was induced by addition of 0.1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) followed by overnight incubation at 15°C and shaking at 220 rpm. Cells were harvested by centrifugation at 4,000 × g for 20 min at 4°C and frozen until use. The bacterial pellet was thawed and resuspended in buffer A [100 mM Tris·HCl (pH 8.3), 30 mM NaCl, 5% glycerol] containing 0.6 mg/ml lysozyme and incubated on ice for 30 min. Cells were disrupted by sonication and insoluble material was pelleted at 245,000 ´g for 1 h at 4°C. The clarified lysate was loaded onto a Ni-NTA column (Qiagen) previously equilibrated with buffer A. The column was washed with buffer A containing 20 mM imidazole and bound proteins were eluted with buffer A containing 200 mM imidazole. Eluted proteins were pooled and loaded immediately onto a Hiload 16/60 Superdex 75 prep grade column (GE Healthcare) previously equilibrated with buffer A. Fractions corresponding to pure MiaE2H were pooled and concentrated using Amicon Ultra-15 centrifugal filter device (Millipore) with a 5 K cut-off. Pure and concentrated MiaE2H was frozen in liquid nitrogen and stored at -80°C until use.

Extinction coefficient was determined by combined optical spectroscopy and amino acid analysis of purified MiaE2H protein (e₂₈₀ = 60,000 M^-1·cm^-1). Samples of known protein concentration were also subjected to metal analysis by atomic absorption spectroscopy. Iron bound-protein was determined according to standard procedure (2).

Because E. coli contains the unhydroxylated ms²i⁶A in its tRNAs, we used the E. coli DH5a strain as the naturally occurring ms²io⁶A-deficient strain to assay the activity of the expressing plasmids of miaE gene in vivo. The E. coli strain was transformed with the expressing plasmids coding for the MiaE1, MiaE2, and MiaE2H proteins and grown in 100 ml of LB medium supplemented with 100 mg/ml ampicillin at 37°C until OD₆₀₀ reached 0.8. tRNAs were isolated as described previously (3), 50-100 mg of purified tRNAs were digested to nucleosides by nuclease P1 and bacterial alkaline phosphatase treatment. The resulting hydrolysate was analyzed by HPLC as described (4, 5).

For the in vitro assay the mixture (100 ml) contained, in 100 mM Tris·HCl (pH 7.5), 50 mM MiaE protein, 50-100 mg of purified tRNAs, and 20 ml of cell-free extracts at 30 mg/ml total proteins.

MiaE2H (50 mM) and the tRNA substrate were combined under the same conditions described above in the absence of cell-free extracts. The reaction was initiated by adding H₂O₂ ( 20 mM). Product identification was as described above.

Aerobic UV-visible absorption spectra were recorded on a Cary 1Bio spectrophotometer (Varian).

EPR spectra of as-isolated and reduced MiaE protein were recorded at X-band by using a Bruker EMX spectrometer equipped with an Oxford ESR 910 cryostat for low temperature studies. For determination of the spin concentration at g = 2 the system was calibrated against a copper-EDTA sample. Spin quantitation of the g = 4.3 resonance was performed at 4 K by using Fe-Desferal as a standard.

. The ⁵⁷Fe Mössbauer experiments were performed by using a 30-mCi (1 Ci = 37 GBq) source of ⁵⁷Co(Rh). The 14.4 keV g-rays were detected by means of a proportional counter, and Mössbauer spectra were recorded on a 512 multichannel analyzer working in multiscaling mode. The system was calibrated with a metallic iron absorber at room temperature, and isomer shifts were reported relative to a-iron at room temperature. The source was maintained at room temperature and moved by a constant acceleration electromechanical drive system under feedback control. Low-field Mössbauer spectroscopy measurements were carried out with homemade axial and transverse magnetic field systems (6). The sample was placed in the tail section of a variable temperature cryostat and maintained at liquid He temperature. It contained ≈1.9 mM ⁵⁷Fe in a 200-ml nylon cell. The spectra were fitted as described (7) with the software package WMOSS (WEB Tesearch, Edina, MN, www.wmoss.org).

HYSCORE experiments were performed on a Bruker Elexsys E-580 X-band pulsed spectrometer with a Bruker ER4118X dielectric resonator and a continuous flow He cryostat (CF935; Oxford Instruments) controlled by an Oxford Instruments temperature controller ITC 503. Experiments were performed at 5 K using the standard four-pulse sequence (p/2-t-p/2-t1-p-t2-p/2-echo) with a nominal pulse width of 16 ns for p/2 and p pulses, a t value of 128 ns and a pulse repetition rate of 2 kHz. Unwanted echoes were removed by four-step phase cycling. A 128 ´ 128 data set was recorded with times t1 and t2 incremented in 20-ns steps from an initial value of 200 ns. This dataset was processed by using Xepr software (Brucker). The background decay in both dimensions was subtracted using a linear fit, followed by apodization with a Hamming window and zero-filling to 512 points in each dimension. The 2D Fourier Transform magnitude spectrum was calculated and presented as a contour plot.

1. Pierrel F, Bjork GR, Fontecave M, Atta M (2002) J Biol Chem 277:13367-13370.

2. Fish WW (1988) Methods Enzymol 158:357-364.

3. Buck M, Connick M, Ames BN (1983) Anal Biochem 129:1-13.

4. Gehrke CW, Kuo KC (1989) J Chromatogr 471:3-36.

5. Gehrke CW, Kuo KC (1990) in Chromatography and Modification of Nucleosides, ed (Elsevier, Amsterdam, The Netherlands), pp A3-A71.

6. Jeandey C, Horner O, Oddou JL, Jeandey C (2003) Measure Sci Technol 14:629-632.

7. Horner O, Mouesca JM, Oddou JL, Jeandey C, Niviere V, Mattioli TA, Mathe C, Fontecave M, Maldivi P, Bonville P, et al. (2004) Biochemistry 43:8815-8825.