An engineered lantipeptide synthetase serves as a general leader peptide-dependent kinase† †Electronic supplementary information (ESI) available: General experimental procedures, primer sequences, and supporting figures. See DOI: 10.1039/c2cc34138g Click here for additional data file.

Mutants of the highly promiscuous lantipeptide synthetase ProcM phosphorylate a wide range of peptides attached to ProcA leader peptides, both in vitro and in E. coli. As such, the ProcM mutants are useful enzymes for the enzymatic preparation of phosphorylated peptides.

primer. The desired PCR product was purified. All other genes encoding the desired peptides were constructed in a similar manner with their corresponding primers in Table S3.
The expression vector (pET15a) and all final PCR products were successively digested with NdeI and XhoI restriction enzymes at 37 °C for 3 h and overnight, respectively. The digested products were purified with the Qiagen PCR cleanup kit and were ligated using Quick T4 DNA ligase for 5 min at room temperature. E. coli DH5α cells were transformed with the ligation mixture and the desired sequences of resultant plasmids were verified by sequencing.
E. coli Rosetta 2 cells were transformed with pET15a expression vectors containing genes encoding various peptides. A single colony was used to inoculate 120 mL of LB media supplemented with 75 μg/mL of ampicillin and incubated at 37 °C with shaking for 14 h. The pre-culture was harvested at 6000 x g at 4 °C for 10 min, the dry pellet was re-suspended in terrific broth (TB) and used to re-inoculate 6 L of TB medium supplemented with 75 μg/mL of ampicillin. The culture was grown at 37 °C to an OD 600 of 0.7. The cells were induced with 1 mM IPTG and incubated for an additional 3 h at 37 °C with shaking. Cells were harvested by centrifugation (6000 x g for 10 min at 4 °C). Pellets were re-suspended in ProcA start buffer (20 mM NaH 2 PO 4 , 500 mM NaCl, 0.5 mM imidazole, 20% glycerol, pH 7.5) and were subjected to sonication cycles of 4 s bursts, 9.9 s rest for a total of 15 min. Lysed cells were centrifuged at 14,000 x g for 30 min at 4 °C and the supernatant was discarded. The remaining pellet was washed with LanA start buffer, sonicated, centrifuged and the supernatant (soluble wash fraction) was discarded similar to the previous step. The pellet was washed and then resuspended in 60 mL of LanA buffer 1 (20 mM NaH 2 PO 4 , 500 mM NaCl, 0.5 mM imidazole, 6 M guanidine, pH 7.5), sonicated, and centrifuged again. The supernatant was applied to a HisTrap column, which was equilibrated with 5 column volumes (CV) of LanA buffer. The bound His-peptide was washed with 5 CV of LanA buffer 1 followed by 5 CV of LanA buffer 2 (20 mM NaH 2 PO 4 , 300 mM NaCl, 30 mM imidazole, 4 M guanidine, pH 7.5). The peptides were eluted from the column with LanA elution buffer (20 mM NaH 2 PO 4 , 100 mM NaCl, 1 M imidazole, 4 M guanidine, pH 7.5) and stored at room temperature for 24-48 h. The Ni-NTA purified peptides were desalted by three different methods depending on scale. The traditional desalting method performed involved preparative reversed-phase HPLC (Waters Delta 600) employing a Waters C4 PrepPak cartridge. The purification was performed at room temperature by applying a linear gradient of 2% solvent A (80% acetonitrile and 0.1% TFA in water) to 100% solvent A over 45 min with the second solvent 0.1% TFA in water (solvent D). The flow rate was set to 8 mL/min and the absorbance at 220 nm was monitored. Fractions containing the desired peptide were pooled and lyophilized (Labconco). The product was analyzed by MALDI-ToF MS and stored at −20 °C. This desalting method was performed with high expressing peptides or cultures over 2 L. A more rapid procedure for relatively high peptide amounts involved precipitation. In this case, the Ni-NTA purified peptides were dialyzed for 2 h at room temperature against sterile deionized water that is 200-500 times the volume of the Ni-NTA eluent. The peptides were moved to fresh sterile deionized water for another 2 h and the water was replaced again and the peptide solution was dialyzed overnight at room temperature. The following morning the precipitated peptide was isolated, solubilized in acetonitrile, and lyophilized. The product was stored at −20 °C after the correct mass was confirmed by MALDI-ToF MS. Peptides that expressed in low quantities or obtained from small cultures were desalted by using C4 solid phase extraction columns (Grace Vydac) as directed by the product manual.

ProcM in vitro phosphorylation assays
Purified lyophilized ProcA peptides (20 μM) were dissolved in water and DMSO (DMSO final concentration <5%) because of solubility issues and were incubated with ProcM T516A (2 μM) in activity buffer (50 mM HEPES, 10 mM MgCl 2 , 5 mM ATP, 1 mM TCEP, pH 7.5) for 5-20 h at room temperature. The reaction was quenched by addition of TFA to a final concentration of 0.5%. The reaction mixtures were subjected to C4 SPE columns to remove ProcM T516A. The C4 SPE eluent was lyophilized and re-suspended in 50 mM Tris, pH 8.0 and Factor Xa, trypsin, LysC or GluC was added and the solution was incubated for an additional 6 h to overnight at room temperature before analysis by MALDI-ToF MS.

Cloning of substrates into ProcM R510M or T516A (MCSII)-pRSFDuet
The genes encoding peptides (Proc-AKT, Proc-CaM, Proc-PKCμ and Proc-PKC) were amplified in the same manner as described above with the primers listed in Table S3. The only difference was that the resultant digested PCR product was ligated into multiple cloning site I (MCSI) of pRSFDuet vectors that already contained ProcM R510M or ProcM T516A in MCSII.

Co-expression and purification of ProcA 2.8 (or ProcM substrates) from ProcM mutants (MCSII)-pRSFDuet
Co-expression of ProcA 2.8 and ProcM (or ProcM mutants) was completed under the same conditions as over-expression of ProcM protein. Purification of the modified ProcA 2.8 (or other modified substrate peptides) was facilitated by a His tag as described above in the purification of ProcA peptides. The resultant peptide was treated with protease and analyzed by MALDI-ToF MS.  Table S2: ProcM kinase substrate sequences. Kinase products were digested at the C-terminus of the red amino acid using LysC or trypsin for cleavage after K and GluC for cleavage after E, and Factor Xa for cleavage after IEGR. Phosphorylations took place at the italic residues within the green region.    ProcM R510M in E. coli, purified by Ni 2+ -NTA affinity chromatography and treated with LysC protease. The same sample was also analyzed by reverse-phase (C18) HPLC. These representative HPLC traces and MALDI-MS spectra show that for the peptides in this study, the MALDI spectra are reasonable indicators of the abundance of phosphorylated and unphosphorylated peptides.
Analysis of phosphorylated products with calf intestine phosphatase. To assure that the mass additions upon treatment with ProcM mutants indeed correspond to phosphorylations, the product peptides were subvjected to two enzymatic assays. Treatment with calf intestine phosphatase resulted in the conversion of the products back to their staring peptides, as expected for phosphorylated peptides ( Figure S7). Furthermore, treatment of the product peptides with wild type ProcM resulted in peptides with masses consistent with phosphate elimination to generate dehydro amino acids (dehydroalanine from Ser, dehydrobutyrine from Thr; Figure S8). Collectively, these observations can only be explained if the products were phosphorylated on Ser/Thr.