Molecular basis for the broad substrate selectivity of a peptide prenyltransferase

Abstract

The cyanobactin prenyltransferases catalyze a series of known or unprecedented reactions on millions of different substrates, with no easily observable recognition motif and exquisite regioselectivity. Here we define the basis of broad substrate tolerance for the otherwise uncharacterized TruF family. We determined the structures of the Tyr-prenylating enzyme PagF, in complex with an isoprenoid donor analog and a panel of linear and macrocyclic peptide substrates. Unexpectedly, the structures reveal a truncated barrel fold, wherein binding of large peptide substrates is necessary to complete a solvent-exposed hydrophobic pocket to form the catalytically competent active site. Kinetic, mutational, chemical, and computational analyses revealed the structural basis of selectivity, showing a small motif within peptide substrates that is sufficient for recognition by the enzyme. Attaching this 2-residue motif to two random peptides results in their isoprenylation by PagF, demonstrating utility as a general biocatalytic platform for modifications on any peptide substrate.RiPP | biosynthesis | prenylation | crystallography

Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis

“…In the case of compounds 5 and 1 + 3 , both the mono-geranylated and un-geranylated peaks were observed because the geranyl group is partially eliminated in the ion source, as has been previously observed in prenylated tyrosine derivatives. 15 (D) Chemical structure of cyc-MSGVDyYNP ( 1 ) showing representative correlations from 2D-NMR spectra. (E) Enzymatic synthesis of MSGVDyYNP ( 5 ).…”

“…We previously reported a crystal structure of the homologous DMAPP prenyltransferase PagF, wherein substrate 9 was in the active site adjacent to a prenyl donor analog. 15 In incubations with 9 and DMAPP, PagF was found to yield predominantly the Tyr1 prenylated analog, with a lower amount of other products. Considering that Tyr 1 was found in proximity to the prenyl donor analog in PagF, we hypothesized that PirF would also predominantly modify the N -terminal Tyn.…”

“…15,17 HPLC and LC-MS analyses demonstrated that PirF catalyzed geranylation of natural and unnatural Tyr ( 7 and 8 ), Tyr-Tyr-Tyr tripeptide ( 9 ), and cyclic and linear Tyr-containing peptides ( 2 and 4 ). Like PagF, a homologous enzyme involved in prenylagaramide B biosynthesis, 15,24 PirF preferred larger peptides to small amino acids.…”

“…15,17 HPLC and LC-MS analyses demonstrated that PirF catalyzed geranylation of natural and unnatural Tyr ( 7 and 8 ), Tyr-Tyr-Tyr tripeptide ( 9 ), and cyclic and linear Tyr-containing peptides ( 2 and 4 ). Like PagF, a homologous enzyme involved in prenylagaramide B biosynthesis, 15,24 PirF preferred larger peptides to small amino acids. Interestingly, small phenolic compounds including 3- or 4-hydroxybenzoic acids ( 13 and 14 ), 2,4- or 3,4- dihydroxybenzoic acids ( 15 and 17 ), and ferulic acid ( 19 ) were also geranylated, while Tyr-derivatives such as L-DOPA ( 24 ) and dopamine ( 25 ) were not.…”

Enzyme-Catalyzed Intramolecular Enantioselective Hydroalkoxylation

“…13 Schmidt et al showed that the forward C-prenylated tyrosine residue in cyanobactins is the product of a nonenzymatic Claisen rearrangement following a reverse O-prenylation catalyzed by the prenyltransferase LynF. 14 However, such rearrangement has not been observed for the prenyltransferases catalyzing forward O-prenylations, such as PagF 15 and SirD. 16 Indeed, density functional theory (DFT) calculations show that Claisen rearrangement after the forward O-prenylation at C7-OH of 5 would be very slow at room temperature (Δ G ‡ 298 = 30.2 kcal/mol with predicted t 1/2 (25 °C) = 4.2 × 10 5 h, Figure S35).…”