Structure Prediction and Genome Mining‐Aided Discovery of the Bacterial C‐Terminal Tryptophan Prenyltransferase PalQ

Abstract Post‐translational prenylations, found in eukaryotic primary metabolites and bacterial secondary metabolites, play crucial roles in biomolecular interactions. Employing genome mining methods combined with AlphaFold2‐based predictions of protein interactions, PalQ , a prenyltransferase responsible for the tryptophan prenylation of RiPPs produced by Paenibacillus alvei, is identified. PalQ differs from cyanobactin prenyltransferases because of its evolutionary relationship to isoprene synthases, which enables PalQ to transfer extended prenyl chains to the indole C3 position. This prenylation introduces structural diversity to the tryptophan side chain and also leads to conformational dynamics in the peptide backbone, attributed to the cis/trans isomerization that arises from the formation of a pyrrolidine ring. Additionally, PalQ exhibited pronounced positional selectivity for the C‐terminal tryptophan. Such enzymatic characteristics offer a toolkit for peptide therapeutic lipidation.

of the samples under the same conditions.Native agarose gel electrophoresis was carried out using a 1.7% agarose gel, in buffer composed of 25 mM Tris and 190 mM glycine (pH 8.3).

Kinetic analysis
The assay solution consisted of 50 mM Tris-HCl, pH 9.0, 150 mM NaCl, 1 mM TCEP, 10% glycerol, 5 mM MgCl2, and a nominal amount of 10 nM PalQ.For the donor substrate kinetics, the reaction mixture was formulated with a range of concentrations (0.01 to 2.5 mM) of the donor substrates; specifically DMAPP, GPP, FPP.This mixture also contained 200 µM of PalX.The mixture designated for acceptor substrate kinetics was composed of 2 mM DMAPP and either PalX at concentrations between 0.005 and 0.25 mM or DNVRRFFW ranging from 0.01 to 2.5 mM.The mixtures were incubated for 4 hours at RT.For the kinetics of DNVRRFFW, the incubation time was changed to 18 hours because of its low reactivity.The reactions were stopped by the addition of EDTA (pH 8.0) to a final concentration of 100 mM.Precipitated protein was removed by centrifugation before the analysis of the reactions by HPLC (Shimadzu).
The HPLC conditions were as follows: the mobile phase consisted of (A) H2O containing 0.1% TFA and (B) acetonitrile containing 0.1% TFA, in a gradient from 80:20 to 20:80 over 10 min.The flow rate was 1.00 mL/min.The column was COSMOSIL 5C18-AR-II (4.6 mm × 100 mm, Nacalai Tesque).The column oven temperature was set at 40 °C and the injection volume was 50 µL.Quantification of both substrate and product was achieved by measuring the absorbance at 260 nm.Kinetic parameters were estimated by fitting the initial velocities to the Michaelis-Menten equation (for DMAPP) or the substrate inhibition equation (for GPP, FPP, NHLP and DNVRRFFW) by a nonlinear least squares method, using the ORIGIN software (OriginLab).

Molecular modeling
The multimeric structures of PalQ and the PalX complex was predicted by AlphaFold2 via ColabFold [5,6] .The magnesium ions were positioned based on the confidence score from the ProBiS server [7] .DMAPP (PDBID: DMA) and FPP (PDBID: FPP) were modeled individually using the "drag coordinates" function within the PyMOL Molecular Graphics System, version 2.3.5 (Schrödinger, LLC).For this modeling, we drew guidance from the X-ray structure of the bacterial polyprenyl synthase complex (PDBID: 3OYR) [8] .The selection of 3OYR as a reference was supported by the structural similarity, as indicated by the DALI server [9] .The CHARMM force field parameters for each isoprenyl diphosphate were created using CGenFF.The PDB files corresponding to each isoprenyl diphosphate were transformed to the mol2 format, and adjusted to pH 7 conditions with the Open Babel software [10] .These mol2 files served as input for CGenFF.
The constructed structures were then subjected to molecular dynamics simulations using GENESIS 1.6.0 [11]These simulations were performed with the Generalized Born/Surface Area (GBSA) implicit solvation model.Production runs were performed for 10 ns at 298.15 K.All bonds inclusive of hydrogen atoms remained constrained during the production run.The time step for integration was set to 2 fs.Temperature and pressure were regulated by the Langevin thermostat.The MD trajectories were analyzed and visualized by VMD [12] .
Molecular modeling of the complex consisting of B. subtilis strain 168 ComQ, ComX, FPP, and Mg 2+ was conducted in a manner similar to that of the PalQ-PalX complex described above.

Section S2: Tables and Figures Table S1
List of homologous enzymes and their putative precursors within the sequence similarity network cluster containing PalQ.ComQ, visualized using ESPript 3.0 [13] .Residues in the first aspartate-rich motif FARM and the (pseudo) second aspartate-rich motif SARM are underlined.S1.

Figure S1 .Figure S2 .
Figure S1.Chemical structures of eukaryotic and bacterial prenylations.Forward prenylations are represented by blue lines, while reverse prenylations are depicted in red.Prenylated residues are highlighted in magenta.X-ray structures of a representative eukaryotic prenyltransferase and a cyanobactin prenyltransferase are shown above.

Figure S3 .
Figure S3.(A) Detailed representation of the P. alvei PalX biosynthetic gene cluster.The postulated functions of neighboring genes are indicated.(B) Gene alignment of the PalX cluster among Paenibacillus xylanivorans, Paenibacillus alvei, and Paenibacillus oryzisoli.Genome IDs for each bacterial species are displayed below their respective names.

FARMFigure S4 .
Figure S4.(A) MALDI-TOF-MS of His-tagged ComX incubated with and without the Paenibacillus alvei cell lysate.The peaks corresponding to His-PalX 2-103 and 2-95 are highlighted in blue and purple, respectively.Peaks indicated by an asterisk (*) could not be assigned based on amino acid sequence information.

Figure S7 .
Figure S7.AlphaFold2 model of the PalQ and PalX complex, with the pLDDT confidence score depicted in red for high-confidence regions.

Figure S8 .B
Figure S8.Size exclusion chromatography (SEC) analysis.(A) SEC analysis of PalQ following His-tag purification revealed an elution volume of 13.19 ml, which corresponds to a molecular weight of 76 kDa.This suggests the formation of a PalQ homodimer.(B) SEC analysis of PalX following His-tag purification.(C) A 1:1 molar mixture of PalX and PalQ was subjected to SEC.Both PalX and PalQ co-eluted in the void volume.(D) The protein fractions from the void volume

Figure S9 .
Figure S9.PalQ activity dependence on donor prenyl diphosphate concentrations.Data were derived from HPLC peak intensities at saturating levels of the acceptor, His-PalX.Kinetic parameters are also provided.For better fitting, we used a substrate inhibition model for GPP and FPP.

Figure S10 .Figure S11. 1 H- 1 H
Figure S10.Dependence of PalQ activity on the concentrations of acceptor substrates.Data were determined using HPLC peak intensities at saturating concentrations of DMAPP.For better fitting, we used a substrate inhibition model.Data are shown as mean ± standard error of the mean.

Figure S17 .
Figure S17.MALDI-TOF-MS spectra of DNVRRFFW after incubations with DMAPP, PalQ and various metal ions.Data were acquired at the endpoints of the prenylation assay.Peaks corresponding to unmodified and mono-prenylated peptides are highlighted in cyan and yellow, respectively.

Figure S18 .
Figure S18.MALDI-TOF-MS spectra of His-tagged PalX following incubation with DMAPP in the presence of either wild-type PalQ or the D200A/D204A mutant.

Figure S19 .
Figure S19.(A) AlphaFold2 model of the ComQ and ComX complex, (B) with the pLDDT confidence scores depicted in red to indicate high-confidence regions.

Figure S20 .
Figure S20.Structures of the active sites of PalQ and ComQ, obtained through MD simulations.Close-up views of the donor FPP binding cavities for PalQ (A) and ComQ (B) are shown with gray surface representations.Close-up views of the acceptor binding surfaces of PalQ (C) and ComQ (D), with the areas within 4.5 Å of the prenylated tryptophan highlighted in orange.

Table S2
List of primers.