Supporting Information. Parsons*, Institute for Bioscience and Biotechnology Research, University of Maryland and National

Transcription

1 Supporting Information Structure of Aminodeoxychorismate Synthase from Stenotrophomonas maltophilia Asim K. Bera, Vesna Atanasova, Anjali Dhanda, Jane E. Ladner,, and James F. Parsons*, Institute for Bioscience and Biotechnology Research, University of Maryland and National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, Maryland

2 Expression and purification of PabA, PabC, and PhzE. E. coli PabC (ADC lyase) and PabA (the ADC synthase glutamine aminotransferase component) were expressed and purified as follows. PabC was purified from cultures of E. coli strain JV30 [ara, (lac-proab), thi, stra, (ø 80 lacz M15), (srl-reca) 306::Tn10 (tet r ) [F : tra D36, proab, laci q Z M15]]. Cells harboring the plasmid pkk233-pabc were grown at 37 C in LB media supplemented with 0.01% vitamin B 6 until A 600 =0.3, at which time the media was made 1 mm in IPTG, and then harvested after an additional 4 hours. About half of the expressed ADC lyase remained soluble under these growth conditions. Purification was performed at 4 C and exposure to light was minimized. The cells were lysed in 100 mm potassium phosphate (ph 7.5), 1 mm EDTA, 1 mm DTT, 0.2 mm PMSF at 9000 psi using a French Press, and cell debris was removed by centrifugation at 20,000xg for 30 min. A 25-45% ammonium sulfate fraction was resuspended in and dialyzed against 20 mm potassium phosphate (ph 6.0), 0.2 mm EDTA, 0.2 mm PMSF. The dialysate was applied to a DEAE Sepharose Fast Flow (Pharmacia) column and eluted with a linear gradient to 1 M KCl. Fractions containing ADC lyase were dialyzed against 20 mm potassium phosphate (ph 7.5) containing 1 mm EDTA and 1 mm DTT and applied to a Reactive Yellow 3 agarose column and eluted with the same buffer containing 1.5 M NaCl. ADC lyase-containing fractions were concentrated and dialyzed against 50 mm potassium phosphate (ph 7.5), containing 1 mm EDTA before application on a TSK-gel G3000SW size exclusion column (TosoHaas). Fractions from gel filtration judged to be pure by SDS-PAGE analysis were pooled, concentrated to ~25 mg/ml and stored at -80 C. E. coli PabA was expressed from a pet20b expression vector in E. coli strain BL21(DE3). Cells were grown at 30 C in LB media. When the A 600 of the culture reached 0.8 OD, IPTG was

3 added to a concentration of 1 mm and the growth was continued for an additional six hours. Cells were harvested by centrifugation and lysed by sonication after resuspension in buffer containing 20 mm Hepes, 1 mm DTT, 1 mm EDTA (ph 8.0). After centrifugation to remove insoluble material, the lysate containing PabA was applied to a HQ50 (Applied Biosystems) anion exchange column. The column was washed with 50 mm Hepes (ph 8.0). PabA was recovered in the flow-through. Fractions containing PabA were pooled, dialyzed against 20 mm Mops, 1 mm DTT (ph 6.5) and applied to a HS20 (Applied Biosystems) cation exchange column. PabA was again recovered in the flow though. Fractions containing PabA were again pooled, concentrated, and applied to a 2.5 cm x 100 cm Superdex 75 (GE) column equilibrated and eluted with 25 mm Tris (ph 7.8), 1 mm DTT, 5% glycerol. Fractions judged pure by SDS- PAGE were pooled and PabA was concentrated to 6.5 mg/ml and stored at -80 C. P. aeruginosa PhzE was expressed from a codon optimized (Genscript) pet28a-phze plasmid in E. coli strain BL21(DE3). Cells were grown initially at 37 C until the culture density reached an optical density of ~0.3 at 600 nm at which time the temperature of the shaking incubator was reduced to 20 C. The bacterial cells were then harvested by centrifugation after h and stored at -20 C until needed. SmPabB was purified by cobalt ion affinity chromatography essentially as directed by the resin manufacturer (Pierce). Human α-thrombin (Haematologic Technologies) was used to remove the portion of the fusion tag encoded by the pet-28a vector. Thrombin was then removed by passing the mixture over benzamidine agarose. The cleaved fusion tag was removed by a second passage over the cobalt resin. PabB judged pure by SDS- PAGE analysis was concentrated to ~10 mg/ml, dialyzed against 50 mm Tris, 50 mm NaCl, 1 mm DTT, 1 mm EDTA (ph 7.8) and stored in 0.2 ml aliquots at -80 C.

4 Figure S1. Superposition of the structures shown in Figure 3 illustrating that residues ~ of SmPabB correspond to the vertical segment of the orange kinked helical segment seen in TrpE and other chorismate-utilizing enzymes. Residues of SmPabB are shown in green here for clarity and the surface of TrpG is not shown. Cartoon diagrams of the TrpEG complex (1I1Q; TrpG shown in red, TrpE shown in gray except residues shown in orange) and SmPabB (blue except residues shown in green). Two β-strands of SmPabB are also peeled away as part of the remodeling. One end of each strand remains in the expected position (marked by asterisks on the corresponding strands of 1I1Q) despite the sheet being twisted out of position.

5 Figure S2. LC/MS analysis for the presence of tryptophan (m/z calcd = ) in purified samples of four chorismate-utilizing enzymes. Extracted-ion chromatograms show that E. coli PabB and SmPabB samples contain tryptophan while PhzE (ADIC synthase) and MenF (isochorismate synthase) samples do not.

6 Figure S3. The dissociation constant for the SmPabB-tryptophan complex was determined by isothermal titration calorimetry. Upper panel, raw data showing the heat exchange following an initial 0.5 µl injection and subsequent 1 µl injections of 2 mm tryptophan into 100 µm SmPabB. Lower panel, plot of reaction enthalpy vs the molar ratio of tryptophan to SmPabB. The data were fit to a single site model and the following parameters were derived: K d = 11.7 ± 3 µm, H = ± 194 cal/mol, S = cal/mol/deg, and n = ±.004. The low stoichiometry is consistent with the observation that we were unable to remove all tryptophan from the protein prior to the ITC experiment. Since SmPabB is monomeric in solution the dissociation constant determined here is not influenced by the partial initial saturation. The initial injection was not included in the fit.

7 Figure S4. LC/MS analysis illustrating that ADIC is not efficiently sequestered by SmPabB. Inclusion of PhzD traps free ADIC and converts it to DHHA (A) UV absorbance chromatogram illustrating that chorismate was converted to either DHHA (0.77 min) or p-aminobenzoate (3.54 min) in the presence of SmPabB, PabA, PabC (15 µm), and PhzD 50 (nm). (B) and (C) are extracted ion chromatograms confirming the identity of each compound. DHHA was further confirmed by subsequent complete conversion to phenazine-1-carboxylate by addition of PhzF, PhzB, and PhzG to the reaction mixture (data not shown; (1, 2)).

8 Figure S5. SmPabB steady-state kinetics using glutamine as the ammonia donor and E. coli PabA as the glutaminase subunit. The variable substrate was chorismate. Kinetic constants are k cat = ±0.005, K m = 237 ± 87 µm.

9 Figure S6. SmPabB steady-state kinetics using glutamine as the ammonia donor and E. coli PabA as the glutaminase subunit. The variable substrate was ADIC. Kinetic constants are k cat = ±0.003, K m = 45 ± 19 µm. (1) Parsons, J. F., Calabrese, K., Eisenstein, E., and Ladner, J. E. (2003) Structure and mechanism of Pseudomonas aeruginosa PhzD, an isochorismatase from the phenazine biosynthetic pathway, Biochemistry 42, (2) Parsons, J. F., Song, F. H., Parsons, L., Calabrese, K., Eisenstein, E., and Ladner, J. E. (2004) Structure and Function of the Phenazine Biosynthesis Protein PhzF from Pseudomonas fluorescens 2-79, Biochemistry 43,