pyridoxal phosphate synthase

Transcription

1 Supplementary Information 13 C-NMR snapshots of the complex reaction coordinate of pyridoxal phosphate synthase Jeremiah W. Hanes, Ivan Keresztes, and Tadhg P. Begley * Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY * To whom correspondence should be addressed at the Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY Tel: (607) tpb2@cornell.edu.

2 Supplementary Figures Supplementary Figure 1 Evaluation of the kinetic competence of Pdx1-Z 1. (a) Pdx1-Z 1 was prepared using 32 P-labeled R5P which was synthesized in situ with γ- 32 P-ATP, D-ribose and ribokinase as described in the methods section. Shown is a 15% SDS-PAGE phosphor-autoradiographic image of the time course of Pi release upon addition of NH 4 Cl to Pdx1-Z 1. (b) The fraction of Pdx1-Z 1 (blue dots) and Pi (black dots) was plotted at a function of time. Prior to plotting, the data were corrected for the small amount of Pi (~ 0.2 per enzyme active site) that was present at time = 0 by subtraction. The Pi present at time = 0 could be due to the fact that Pdx1 is able to bind Pi in a site distinct from the R5P binding site as seen in the crystal structure. 1 (c) The absorbance at 320 nm is plotted as a function of time for a sample prepared in an identical to that shown in (a) and (b) except that non-radioactively labeled ATP was used in the in situ preparation of the R5P. From a fit of the data using non-linear regression to a single exponential equation, the rate of accumulation of both Pi and absorbance at 320 nm were within experimental error (0.062 ± min -1 and ± min -1, respectively).

3 Supplementary Figure 2 13 C-dqfCOSY spectrum of [ 14 N]-Pdx1-Z 1. The double quantum filtered COSY was acquired with a sweep width of 30 khz in both dimensions. 256 points were collected in the indirectly detected dimension with 64 scans and 8192 points per increment. The resulting matrix was zero filled to 4096 x 4096 complex data points and shifted sine bell window functions were applied in both dimensions prior to Fourier transformation. (a) Full spectrum (b) Zoom in of spectrum where C1, C3, C4 and C5 are present.

4 Supplementary Figure 3 1 H- 13 C-HSQC spectrum of [ 14 N]-Pdx1-Z 1. The adiabatic HSQC shown above was acquired with a sweep width of 8.4 khz in F2 and 30.1 in F points were collected in the indirectly detected dimension with 4 scans and 2518 points per increment. The resulting matrix was zero filled to 4096 x 4096 complex data points and sine bell window functions were applied in both dimensions prior to Fourier transformation. The numbers in bold are the chemical shift values for each proton and the other values are the carbon chemical shift values.

5 Supplementary Figure 4 13 C spectra of Pdx1-Z 1. (a) Comparison of 15 N Pdx1-Z 1 (top) with Pdx1-Z 1 which contains natural abundance 15 N. (b) C1 region of the 13 C spectrum of Pdx1-Z 1. Pdx1-Z 1 was prepared using 15 N labeled protein and singly C1 labeled D-ribose and broadband 15 N decoupling was applied. A coupling constant of 7.4 Hz was measured. Prior to Fourier transformation, a shifted sine bell window function was applied to the data for resolution enhancement.

6 Supplementary Figure 5 13 C spectra of Pdx1-Z 2, carbons 1, 2, and 5. The data and arrows are color coded and correlate with the following carbon atoms: green is C1, red is C2 and blue is C5. The sample constituents are listed to the right of the spectra along with the broadband 15 N decoupling status. The top two are the same sample without and with 15 N decoupling applied to show that C1, C2 and C5 are coupled to 15 N atoms derived from the [ 15 N]-Pdx1 and the [ 15 N]-NH4Cl used to reconstitute I 320. The lower sample was the same except that [ 14 N]-NH 4 Cl was used to reconstitute I 320. No C-N coupling is observed for the signal corresponding to C2 in this sample indicating that the C-N coupling apparent at this position in the upper sample was due to the incorporation of 15 N from NH 4 Cl. A shifted sine bell function was applied to all the data to enhance the resolution.

7 Supplementary Figure 6 13 C spectra of Pdx1-Z 2, carbons 3 and 4. Pdx1-Z 2 was prepared using universally 13 C labeled D-ribose and both 15 N labeled NH 4 Cl and Pdx1. Shown are the regions corresponding to the C3 and C4 carbons. In the top spectrum, 15 N decoupling was not applied whereas it was applied in the bottom spectrum. Prior to Fourier transformation, a shifted sine bell window function was applied to the data for resolution enhancement.

8 Supplementary Figure 7 Arrayed 15 N decoupling analysis of 13 C spectra for Pdx1-Z 2. Shown are the regions corresponding to the C3 (black), C5 (blue) and C1 (green). The signal representing C3 was included only as a reference for the variation expected due to random noise. Narrowband (Waltz) 15 N decoupling was arrayed in 11.5 ppm increments. Prior to Fourier transformation, a shifted sine bell window function was applied to the data for resolution enhancement.

9 Supplementary Figure 8 Conversion of I 320 to Pdx1-Z 3. I 320 was prepared according to the methods section and was purified from excess small molecules, then a final concentration of 1.5 mm G3P (racemic mixture) was added to start the reaction. (a) Shown are UV-visible scans taken as a function of time. The direction of change over time is illustrated according to the direction of the arrows. (b) The absorbance at 320 nm (blue dots) and 408 nm (red dots) is plotted as a function of time. The rate of decay of I 320 (0.030 ± min -1 ) and the rate of formation of Pdx1-Z 3 (0.030 ± min -1 ) were determined by fitting the data by non-linear regression to a single exponential equation as described in the methods section.

10 Supplementary Figure 9 13 C-dqfCOSY spectrum of 15 N labeled Pdx1-Z 3. The double quantum filtered COSY was acquired with a sweep width of 30 khz in both dimensions. 256 points were collected in the indirectly detected dimension with 64 scans and 8192 points per increment. The resulting matrix was zero filled to 4096 x 4096 complex data points and shifted sine bell window functions were applied in both dimensions prior to Fourier transformation. (a) Full spectrum (b) Zoom in of spectrum where C2, C3 and C4 are present.

11 Supplementary Methods Over-expression of Pdx1 The minimal media recipe for the over-expression of Pdx1 using a total culture volume of 6L was based upon standard M9 minimal media formulations and the amount of each component was as follows: 40.7 g Na 2 HPO 4 18 g KH 2 PO 4 3 g NaCl 6 g NH 4 Cl or [ 15 N]-NH 4 Cl 12 ml of 1M MgSO ml of 20% glucose 1.2 ml 1M CaCl 2 Pdx1-Z 1 preparation The reaction mixture given below was prepared to obtain a total volume of 2 ml of Pdx1-Z 1. Pdx1-Z 2 and Pdx1-Z 3 were derived from Pdx1-Z 1 and were therefore initially composed similarly. The various procedures for the chemical quenching and NMR analysis of the samples are detailed in the main text according to the particular experimental design. Component added Stock concentration Amount added (μl) Pdx1 stock 2 mm 600 HEPES buffer 250 mm HEPES 0.5 M NaCl 288 TCEP 250 mm 16 D-ribose 10 mg/ml Ribokinase 8 mg/ml 19.2 MgCl 2 1 M 9.6 ATP 100 mm ph RT 96 NaCl 4 M 35 H 2 O Double distilled 768 Reference List 1. Zein,F. et al. Structural insights into the mechanism of the PLP synthase holoenzyme from Thermotoga maritima. Biochemistry 45, (2006).