Supplementary Material for. Chasper Puorger, Salvatore Di Girolamo, Georg Lipps *

Transcription

1 Supplementary Material for Elucidation of the Recognition Sequence of Sortase B from Bacillus anthracis by Using a Newly Developed Liquid Chromatography-Mass Spectrometry-Based hod Chasper Puorger, Salvatore Di Girolamo, Georg Lipps * University of Applied Sciences and Arts, Institute for Chemistry and Bioanalytics, Gründenstrasse 4, 4132 Muttenz, Switzerland To whom correspondence should be addressed: georg.lipps@fhnw.ch S 1

2 Table S1: Comparison of esasrta substrate profile at position 2 from different studies this study % substrate conversion Dorr et al., 214 LAETG 8 3 LGETG < 1 - L(I/L)ETG < 1 - LMETG 2 - LPETG LSETG 1 1 S 2

3 Absorbance at 497 nm Figure S M esasrta, 24h M esasrta, 6h M esasrta, 3h 1 5 M esasrta, 1h no enzyme Retention time (min) Figure S1. Time dependence of the transpeptidation reaction of LPXTGE peptide with esasrta. esasrta catalyzed reactions of LPXTGE with GGGK-biotin peptide were stopped after different time-points by acidification and analyzed by RP-HPLC. The three initial substrate peaks (gray chromatogram) decrease over time in intensity as the peptides are consumed in the reaction. Additional peaks appear after 1 h of incubation which are shifted by minutes compared to the corresponding substrate peaks (dashed lines). Peptides in these peaks were determined to be transpeptidation products. Peaks appearing after 3 hours of incubation contain hydrolysis products as was shown with reactions in absence of nucleophile (see Figure S2). S 3

4 Absorbance at 497 nm Figure S M esasrta, 24h M esasrta, 6h M esasrta, 3h 1 5 M esasrta, 1h Retention time (min) no enzyme Figure S2. Time dependence of the hydrolysis reaction of LPXTGE peptide with esasrta. esasrta catalyzed reactions of LPXTGE without nucleophile were stopped after different time -points by acidification and analyzed by RP-HPLC. The three initial substrate peaks (gray chromatogram) decrease over time in intensity as the peptides are consumed in the reaction. Additional peaks appear after 1 h of incubation which are shifted by less than one minute compared to the corresponding substrate peaks. Peptides in these peaks are hydrolysis products. Dashed lines indicate the positions of the peaks with transpeptidation products (see Figure S1). S 4

5 Peak area (mau*min) Figure S Reaction time (h) Figure S3. Analysis of time dependence of transpeptidation product (blue) and hydrolysis product (red) concentrations in the SaSrtA-catalyzed reaction of LPXTGE peptide with GGGK-biotin nucleophile. Transpeptidation product concentration is maximal after 1 h of reaction, while no significant hydrolysis is observed at this time point. With longer incubation hydrolysis products are formed and transpeptidation products (which are again a substrate for esasrta) further decrease slowly. S 5

6 % substrate consumed Figure S4 a b XPETGGGK-bio LXETGGGK-bio c d e LPXTGGGK-bio LPEXGGGK-bio LPETXE Figure S4. Detailed view on the substrate specificity of esasrta. The percentages of product with a specific sequence compared to total formed product of the reactions with substrates XPETGE (a), LXETGE (b), LPXTGE (c), LPEXGE (d) or the percentage of consumed substrate compared to total substrate LPETXE consumption (e) are shown. X represents any of the 2 amino acids at the respective position. Peptide mixtures LXETGE and LPEXGE lacked the peptides with a at position X, as was found by LC-MS analysis of the substrate mixtures. S 6

7 A comparison of our results for the sequence specificity of esasrta with previous results for the same sortase 1 and for wild type SaSrtA 2 shows good correlation. Unfortunately only positions two and four of the recognition sequence where previously analyzed for esasrta allowing a direct compari son with our data. For position two we found predominantly proline in the recognition sequence, with lower occurrence of alanine and serine, as was published by Dorr et al.. 1 However, with leucine/isoleucine, methionine and glycine we found three additional residues which are also recognized by esasrta to a minor extent. At position four we found comparable preferences for serine and threonine, while previous data showed a more pronounced preference for threonine. Other residues which were found at lower abundance in our study and by Dorr et al. are alanine, leucine/isoleucine, glycine and valine. We did however not find any other residues for position four in contrast to previous data where aspartate, histidine and tryptophan were found at low abundance. teine was previously found to be the second most abundant residue, which we could not confirm because the substrate mixture used did not contain a peptide with a cysteine residue at position four. We next compared the substrate specificities of wild type SaSrtA and esasrta. Although esasrta was evolved by mutagenesis the sequence specificities are very similar. At position one the two amino acids leucine and methionine were found for wild type SaSrtA with an about 4 -fold higher preference for leucine. For esasrta we observed predominantly leucine/isoleucine with over 9 % abundance, followed by methionine (5.5 %), proline (3 %) and to a low extent phenylalanine, asparagine, glutamine, valine and tryptophan. For the second position wild type SaSrtA recognizes mainly peptides with proline and t o a minor extent alanine, while we found additional permitted residues for this position (see above). At the third position no clear preference is observed for both wild type SaSrtA and esasrta. The differences between the individual amino acid substitutions seem to be larger for esasrta compared to wild type SaSrtA, this can however possibly be attributed to the difference in reaction time and thus if the end point of the reaction is reached or not (see also 2 ). Analysis of position four again shows similar sequence specificities for the two sortases, with the sole difference that serine is less pronounced in the wild type SaSrtA transpeptidation products compared to the esasrta products. Finally, for position five both sortases seem to permit glycine as sole residue in the recognition sequence. S 7

8 Absorbance at 497 nm Figure S M BaSrtB, 24 h 8 M BaSrtB, 6 h 6 M BaSrtB, 3 h 4 M BaSrtB, 2 h 2 M BaSrtB, 1 h Retention time (min) M BaSrtB, h Figure S5. Time dependence of the transpeptidation reaction of DNPXTGDE peptide with BaSrtB. BaSrtB catalyzed reactions of DNPXTGDE with GGGK-biotin peptide were performed at 25 C and stopped after different time-points by shock-freezing. Transpeptidation products were isolated with magnetic streptavidin beads and analyzed with RP-HPLC. S 8

9 a Figure S b DXPNTGGGK-bio DNXNTGGGK-bio c d DNPXTGGGK-bio DNPNXGGGK-bio e XNPNTGGGK-bio Figure S6. Detailed view on substrate specificity of BaSrtB. The percentages of product with a specific sequence compared to total formed product of the reactions with substrates DXPNTGDE (a), DNXNTGDE (b), DNPXTGDE (c), DNPNXGDE (d) and XNPNTGDE (e) are shown. X represents any of the 2 amino acids at the respective position. S 9

10 References (1) Dorr, B. M., Ham, H. O., An, C., Chaikof, E. L., and Liu, D. R. (214) Reprogramming the specificity of sortase enzymes. c. Natl. Acad. Sci. U. S. A 111, (2) Kruger, R. G., Otvos, B., Frankel, B. A., Bentley, M., Dostal, P., and McCafferty, D. G. (24) Analysis of the substrate specificity of the Staphylococcus aureus sortase transpeptidase SrtA. Biochemistry 43, S 1