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Molecular Basis for the Broad Substrate Selectivity of a Peptide Prenyltransferase

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Molecular Basis for the Broad Substrate Selectivity of a Peptide Prenyltransferase Molecular basis for the broad substrate selectivity of a peptide prenyltransferase Yue Haoa,1, Elizabeth Pierceb,1, Daniel Roeb, Maho Moritab, John A. McIntoshb, Vinayak Agarwala,c, Thomas E. Cheatham IIIb, Eric W. Schmidtb,2, and Satish K. Naira,c,d,2 aDepartment of Biochemistry, University of Illinois at Urbana–Champaign, Urbana, IL 61801; bDepartment of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112; cCenter for Biophysics and Computational Biology, University of Illinois at Urbana–Champaign, Urbana, IL 61801; and dInstitute for Genomic Biology, University of Illinois at Urbana–Champaign, Urbana, IL 61801 Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved October 20, 2016 (received for review June 17, 2016) The cyanobactin prenyltransferases catalyze a series of known or geranyl/farnesyl transferase ComQ, which is involved in quorum- unprecedented reactions on millions of different substrates, with no sensing pheromone production (12, 21, 22), and the cyanobactin easily observable recognition motif and exquisite regioselectivity. enzymes described here, which catalyze the posttranslational side Here we define the basis of broad substrate tolerance for the chain prenylation of peptide natural products (23). otherwise uncharacterized TruF family. We determined the struc- Bioinformatics analysis of metagenome-derived cyanobactin tures of the Tyr-prenylating enzyme PagF, in complex with an biosynthetic clusters led to the proposal of the TruF family of isoprenoid donor analog and a panel of linear and macrocyclic proteins as candidate prenyltransferases (Fig. 1A and SI Appendix, peptide substrates. Unexpectedly, the structures reveal a truncated Fig. S1) (5). Biochemical characterization of LynF (from Lyngbya barrel fold, wherein binding of large peptide substrates is necessary aestuarii) established the TruF family as broadly substrate-tolerant to complete a solvent-exposed hydrophobic pocket to form the O-prenyltransferases that catalyze electrophilic alkylation on Tyr catalytically competent active site. Kinetic, mutational, chemical, residues on cyanobactins, using the five-carbon dimethylallyl py- and computational analyses revealed the structural basis of selec- rophosphate (DMAPP) as the donor (23). Homologs of lynF and tivity, showing a small motif within peptide substrates that is truF are prevalent in nearly all cyanobactin biosynthetic clusters sufficient for recognition by the enzyme. Attaching this 2-residue and can afford both forward- and reverse-prenylated products motif to two random peptides results in their isoprenylation by PagF, (Fig. 1B). Surprisingly, F enzymes are found even in clusters that demonstrating utility as a general biocatalytic platform for modifi- do not produce a prenylated product (24); for example, the cations on any peptide substrate. patellamides are not prenylated, but a patF gene is clearly distin- guishable in the biosynthetic cluster (25). The crystal structure of RiPP | biosynthesis | prenylation | crystallography unliganded PatF reveals a prenyltransferase barrel fold (26), but this enzyme does not catalyze any biochemical reactions. he rational biosynthesis of drug-like small molecules requires a Analysis of prenylation by LynF reveals that the enzyme uses robust set of intrinsically broadly substrate-tolerant enzymes that the mature cyclic peptide as the preferred substrate (23). In other T BIOCHEMISTRY can catalyze diverse chemical reactions. Enzymes that modify pep- cyanobactin pathway enzymes, substrate permissiveness has been tides and proteins are of exceptional interest in drug discovery ef- explained in part by the presence of conserved recognition se- forts, and cyanobactin biosynthetic enzymes have been applied to the quences in the leader peptide, which subsequently are proteolytically creation of chemically diverse analogs, including libraries encoding cleaved during the course of macrocyclization. However, be- potentially millions of prenylated compounds (1). Cyanobactins (2–5) cause prenylation occurs after removal of the leader peptide, compose a family of ribosomally synthesized and posttranslationally there is no obvious recognition element in the sequences of modified peptides (RiPPs) (6), most of which are macrocyclized the hundreds of prenylated variants identified in cyanobactin by transamidation of their N and C termini. A significant subset of pathways. Finally, LynF was able to prenylate several other non- cyanobactins is modified by either forward or reverse isoprenylation natural substrates, including N-acetyl– and N-boc–modified D-or on Ser, Thr, or Tyr residues of the macrocycles, or in some cases on L-Tyr, as well as several other linear and cyclic polypeptides (23). the terminal nitrogen of linear peptides (7). The basis of relaxed substrate specificity has been examined for a series of RiPP Significance modification enzymes, revealing that conserved recognition se- quences within the leader region of the precursor peptide enable The cyanobactin prenyltransferases serve as a tool kit for regio- modification of hypervariable core peptides (8–11). selective and chemoselective peptide and protein modifications, In addition to playing fundamental roles in numerous cellular in which each enzyme can catalyze the same chemistry on an processes, including signal transduction and trafficking (12, 13), enormous number of different substrates. Installation of a mini- prenylation on small-molecule substrates is critical to bioactivity, mal motif is sufficient to direct modifications on any peptide and also contributes to the structural diversity of medically rele- substrate, which can alter their properties to be more drug-like. vant secondary metabolites (14–16). Prenylation directs protein substrates to cell membranes and enhances the ATP-independent Author contributions: T.E.C., E.W.S., and S.K.N. designed research; Y.H., E.P., J.A.M., V.A., cell permeability of peptide substrates (17). Consequently, isoprene T.E.C., and S.K.N. performed research; D.R., M.M., J.A.M., and V.A. contributed new attachment is of great interest as a modification that can increase reagents/analytic tools; Y.H., E.P., D.R., T.E.C., E.W.S., and S.K.N. analyzed data; and cellular uptake (18, 19). Bare peptides are generally considered E.P., D.R., T.E.C., E.W.S., and S.K.N. wrote the paper. poor drug candidates, and lipid attachment improves necessary The authors declare no conflict of interest. properties, such as the ability to penetrate the blood–brain barrier This article is a PNAS Direct Submission. and absorption, distribution, and metabolism (20). Enzymatic Data deposition: The crystallography, atomic coordinates, and structure factors have been strategies for prenylation are well described in higher eukaryotes deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 5TTY (native PagF), 5TU4 (PagF-Boc-Tyr), 5TU5 (PagF-(Tyr)3), and 5TU6 (PagF-cyclic[INPYLYP])]. and include the modification of protein substrates with either a C15 1 or C20 isoprenoid catalyzed by protein farnesyl transferases and Y.H. and E.P. contributed equally to this work. geranylgeranyl transferases, respectively, or the membrane- 2To whom correspondence may be addressed. Email: [email protected] or [email protected]. integral CaaX proteases (12). There are only limited examples This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of bacterial peptide prenyltransferases, including the indole 1073/pnas.1609869113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1609869113 PNAS | December 6, 2016 | vol. 113 | no. 49 | 14037–14042 Downloaded by guest on October 1, 2021 also found in the natural product isolated from the cyanobacteria O. aghardii. Moreover, the Tyr residue in all substrates was modified regioselectively only in the forward direction on the phenolic oxygen. This is similar to what was found for LynF, where only phenolic reverse O-prenylation was observed in a large series of substrates (23). Thus, the cyanobacterial F family enzymes are distinct from canonical small-molecule prenyltransferases for their preference of amino acid substrates only within linear or cyclic peptides, as well as for their strict regioselectivity. Cocrystal Structures of Substrates with PagF. To delineate the de- terminants for substrate recognition, we solved the crystal struc- ture of native PagF (to 1.8 Å resolution), as well as ternary complex structures with the the prenyl donor analog dimethylallyl S-thiolodiphosphate (DMSPP) and boc-L-Tyr (2.1 Å resolution), a Tyr-Tyr-Tyr tripeptide (1.9 Å resolution), and cyclic[INPYLYP] peptide (2.2 Å resolution), the latter of which is one of the phys- iological substrates for this enzyme. Relevant data collection and Fig. 1. (A) Chemical structures of representative cyanobactins highlighting refinement statistics are provided in SI Appendix,TableS3.The relevant chemical features including azoles/azolines (yellow/cyan) installed by overall structure of PagF consists of 10 antiparallel β-strands sur- heterocyclases and isoprenylation (pink) catalyzed by F enzymes. (B)Forward rounded by 10 α-helices to form a barrel architecture reminiscent and reverse prenylation on Tyr residues in substrates. of that observed for aromatic prenyltransferases of the ABBA and indole PTase families of enzymes (Fig. 2A) (14, 16). The DMSPP analog is bound at the center of the barrel, where residues Arg65, The broad substrate tolerance of F-type prenyltransferases Lys136, His138, Tyr186, Tyr235, Arg288, and Tyr290 coordinate
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