Supplemental data
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- Table S2. Data collection and crystallographic refinement statistics
- Crystallographic refinement
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Supplemental Procedures and Data (Pitsawong et al.) Experimental Procedures Reduction Potential Measurement Measurements of reduction potential values (Eo'm) of the mutant enzymes were carried out according to the protocol described in (1), using xanthine/xanthine oxidase as a reduction system. Reactions for all mutants were carried out in reaction mixtures containing 500 µM xanthine, 3 nM xanthine oxidase (side-arm), standard dye (26.8 µM indigo carmine, 18 µM cresyl violet, or 20 µM methylene blue) and Thr169 mutants (26.8 µM T169S; 26.8 µM T169N; 18 µM T169A; or 20 µM T169G) in 50 mM sodium phosphate (pH 7.0). The reaction was placed in an anaerobic cuvette, and was made anaerobic using either an anaerobic glovebox or an anaerobic train (2). After anaerobiosis had been established, a spectrum of the reaction mixture in the oxidized state was recorded before the reaction was initiated by adding xanthine oxidase from the side arm. Reductions of T169S and T169N mutants were monitored using indigo carmine (Eo'm = –116 mV) as the standard dye, similar to the procedure used for the wild-type enzyme (3). Redox potentials of T169A and T169G were determined according to the protocol described above, but using cresyl violet acetate (Eo'm = –166 mV) and methylene blue (Eo'm = +10 mV) as standard dyes, respectively. The Eo'm values were calculated using the standard Nernst equation (1, 4). Rapid-Reaction Experiments – Reductive and oxidative half-reactions of the Thr169 variants were studied using a stopped-flow spectrophotometer (Hi-Tech Scientific). The flow system was made anaerobic by rinsing with an anaerobic buffer and incubating overnight with a solution of 400 μM protocatechuic acid (PCA) and 0.1 Unit/mL of protocatechuic acid dioxygenase (PCD) in potassium phosphate pH 7.0 (5). Prior to the experiment, the instrument was rinsed carefully with anaerobic buffer of 50 mM sodium phosphate equilibrated with N2 gas that had been passed through an oxygen removal column (Labclear). All enzyme solutions were prepared in 50 mM sodium phosphate (pH 7.0), and stopped-flow experiments were performed at 4 or 25 °C. To study the reduction of mutant enzymes by D-Glc or D-Gal, the enzyme solution was placed in a glass tonometer and made anaerobic using an anaerobic glove-box or anaerobic gas train. All buffer and substrate solutions were made anaerobic by bubbling with N2 gas for 6 min, and then loaded onto the stopped-flow machine. Solutions of D-Glc and D-Gal were bubbled with oxygen-free N2 gas for 5 min before being loaded onto the stopped-flow machine. The reduction of the enzyme-bound FAD was monitored at 395 nm and 458 nm. For studying the oxidative half-reaction, solutions containing the reduced enzyme were prepared as described previously (2, 6). In brief, the oxidized enzyme was equilibrated inside the anaerobic glovebox (oxygen < 3 ppm), and then reduced by adding a stoichiometric amount of D-Glc. To ensure complete reduction, the enzyme spectra were monitored using a spectrophotometer inside the glovebox. The reduced enzyme solution was then mixed with buffers equilibrated with oxygen ([O2] = 0.13, 0.31, 0.61, and 0.96 mM) using the stopped-flow spectrophotometer. The reactions were performed in 50 mM sodium phosphate (pH 7.0), and monitored at 5 nm intervals in the range between 300 nm to 550 nm. Crystallographic Analysis – Expression and purification of the T169 mutants used for crystallization have been reported previously (7). For all proteins, crystals grew from microseeded hanging-drops containing a 1:1 mixture of 0.1 M Mes (pH 5.2), 50 mM MgCl2, 10% (w/v) monomethyl ether PEG 2000 and protein at a concentration of 20 mg/mL in 20 mM Mes (pH 5.2). Crystals were stabilized in mother liquor with 28 (w/v) % PEG 2000, followed by vitrification in liquid nitrogen. Data were collected at 100 K using synchrotron radiation at beam line I911-2, MAX-lab, Lund, Sweden, and processed and scaled using the XDS package (8). Phases were obtained either by means of molecular replacement (PHASER; (9)), or Fourier synthesis (FFT, CCP4; (10)) using the refined model of P2O variant H167A as a starting model (PDB code 2IGO; (3)). Crystallographic refinement was performed with REFMAC5 (11), including anisotropic scaling, calculated hydrogen scattering from riding hydrogen atoms, and atomic displacement parameter refinement using the translation, libration, screw-rotation (TLS) model. The TLS models, comprising five to ten TLS groups, were determined using the TLS Motion Determination server (TLSMD; (12)). Model rebuilding was done manually with the guidance of A-weighted 2Fo-Fc and Fo-Fc electron-density maps. The same set of Rfree reflections was used throughout all refinement. No NCS restraints/constraints were used. Model building, coordinate manipulation and least-squares comparisons were made with the program O (13) and Coot (14). Figures were prepared using PyMOL (15). Data collection and refinement statistics are given in Supplementary Table S2. Results 
Fig. 1 Redox potential determination of T169N mutant enzyme using indigo carmine as the standard dye. The reaction contained 500 µM xanthine, 3 nM xanthine oxidase (side arm), 26.8 µM T169N mutant and 26.8 µM indigo carmine. Enzyme and dye were slowly reduced under anaerobic conditions by the xanthine/xanthine oxidase system. The top spectrum is that of a mixture of the fully oxidized enzyme and dye, and the bottom absorption spectrum is that of the fully reduced enzyme and dye. The inset shows a plot of log(Ered/Eox) versus log (Dred/Dox). The slope of the plot of log(Ered/Eox) versus log (Dred/Dox) is 1.2, indicating that two electrons are involved in the reduction of enzyme and dye. The redox potential (Eo'm) for T169N is –95 mV.  
 
Fig. 2 Steady-state kinetics of Thr169 mutants with D-Glc or D-Gal. The assay reactions were performed at 4°C (for D-Glc) and 25 oC (for D-Gal) using the stopped-flow spectrophotometer. Double reciprocal plots of initial rates of T169S (A) and T169G (B) with final D-Glc concentrations of 0.1, 0.2, 0.5, 1, 2, 4, 8, and 12.5 mM. The upper to lower lines are according to final oxygen concentrations of 0.13, 0.26, 0.44, 0.74, and 1.09 mM. Double reciprocal plots of initial rates of T169S (C) with final D-Gal concentrations of 4, 8, 16, 32, 64, and 150 mM, and T169G (D) with final D-Gal concentrations of 1, 2, 4, 8, 16, and 25 mM. The upper to lower lines are according to the final oxygen concentrations of 0.13, 0.26, 0.44, and 0.74 mM. 
Fig. 3 Reduction of the oxidized T169N, T169S and T169G with D-Gal. Stopped-flow traces at 458 nm are shown in dotted lines while at 395 nm are in solid lines. A) A solution of the T169N (23 μM) was mixed with various concentrations of D-Gal (0.8, 1.6, 3.2, 6.4, 15, 30 and 60 mM). All concentrations are given as after mixing. The inset A shows a plot of kobs of the first phase (absorbance increase at 395 nm) versus D-Gal concentrations while kobs of the second phase (large decrease in A458 nm) was plotted against D-Gal concentration in inset B. B) The reduction of T169S by D-Gal was carried out as described in A) and similar plots were shown in the insets. C) The reduction of T169G by D-Gal was carried out as described in A) and similar plots are shown in the insets. Table S1. Nucleotide sequences of synthetic oligonucleotides
Table S2. Data collection and crystallographic refinement statistics
1The outer shell statistics of the reflections are given in soft brackets. Shells were selected as defined in XDS (8) by the user. 2 Rsym = [ hkl i |I–| /hkl i |I| ] x 100 %. 3 Rfactor = hkl | |Fo|–|Fc| | / hkl |Fo| 4 As determined by MolProbity (16). REFERENCES 1. Massey, V. (1991) A simple method for determination of redox potentials, in Flavins and Flavoproteins (Curti, B., Ronchi, S., and Zanetti, G., eds.), Walter de Gruyter, Berlin, pp. 59–66 2. Prongjit, M., Sucharitakul, J., Wongnate, T., Haltrich, D., and Chaiyen, P. (2009) Biochemistry. 48, 4170-4180 3. Kujawa, M., Ebner, H., Leitner, C., Hallberg, B. M., Prongjit, M., Sucharitakul, J., Ludwig, R., Rudsander, U., Peterbauer, C., Chaiyen, P., Haltrich, D., and Divne C. (2006) J. Biol. Chem. 281, 35104-35115 4. Chaiyen, P., Brissette, P., Ballou, D. P., and Massey, V. (1997) Biochemistry.36, 2612-2621 5. Patil, P. V., and Ballou, D. P. (2000) Anal. Biochem. 286, 187-192 6. Sucharitakul, J., Prongjit, M., Haltrich, D., and Chaiyen, P. (2008) Biochemistry. 47, 8485-8490 7. Spadiut, O., Leitner, C., Tan, T. C., Ludwig, R., Divne, C., and Haltrich, D. (2008) Biocatal. Biotran. 26, 120-127 8. Kabsch, W. (1993) J. Appl. Cryst. 26, 795-800 9. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007). Phaser crystallographic software. J. Appl. Cryst. 40, 658-674 10. Collaborative Computational Project, Number 4. 1994. "The CCP4 Suite: Programs for Protein Crystallography". Acta Crystallog. Sect D. 50, 760-763 11. Murshudov, G. N., Vagin, A. A., and Dodson E. J. (1997) Acta Crystallog. Sect. D. 53, 240-255 12. Painter, J., and Merritt, E. A. (2006) Acta Crystallog. Sect. D, 62, 439-450 13. Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallog. Sect. A. 47, 110-119 14. Emsley, P., and Cowtan, K. (2004) Acta Crystallog. Sect. D. 60, 2126-2132 15. DeLano, W.L. (2002) The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA, USA. http://www.pymol.org 16. Lovell, S. C., Davis, I. W., Arendall III, W. B., de Bakker, P. I. W., Word, J. M., Prisant, M. G., Richardson, J. S., Richardson, D. C. (2003) Prot. Struct. Funct. Genet. 50, 437-450 Yüklə 1,14 Mb. Dostları ilə paylaş:
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