Follow the leader: the use of leader peptides to guide natural product biosynthesis

Abstract

The avalanche of genomic information in the past decade has revealed that natural product biosynthesis using the ribosomal machinery is much more widespread than originally anticipated. Nearly all of these compounds are crafted through post-translational modifications of a larger precursor peptide that often contains the marching orders for the biosynthetic enzymes. We review here the available information for how the peptide sequences in the precursors govern the post-translational tailoring processes for several classes of natural products. In addition, we highlight the great potential these leader peptide-directed biosynthetic systems offer for engineering conformationally restrained and pharmacophore-rich products with structural diversity that greatly expands the proteinogenic repertoire.

Figure 1. General Scheme and Examples of Leader Peptide Directed Biosynthesis (LDB)

The precursor peptides typically consist of an N-terminal leader and a C-terminal core peptide. A signal peptide governing subcellular localization may be attached to the N-terminus of the leader peptide, and recognition sequences are sometimes found to the C-terminus of the core peptide. The precursor peptides are ribosomally synthesized and posttranslationally modified to their active structures.

Figure 2. Posttranslational modifications in lantibiotics

a. Different PTMs found in lantibiotics. Colors denote the amino acid origins: red structures are derived from Ser, green from Thr, blue from Cys, and violet from Lys. b. Biosynthesis of lacticin 481, a class II lantibiotic. The lacticin 481 precursor peptide (LctA) containing an N-terminal leader and C-terminal core peptide is transformed into a polycyclic thioether product through the action of a bifunctional enzyme (LctM) that dehydrates Ser to dehydroalanine and Thr to dehydrobutyrine, and subsequently catalyzes the Michael-type addition of Cys residues to these unsaturated amino acids. The leader is proteolytically removed from the modified core peptide by a bifunctional protease/transporter enzyme (LctT). Coloring scheme as in Figure 2a. Although the process is drawn as complete dehydration before the commencement of cyclization, recent studies suggest the dehydration and cyclization events may be alternating^,. c. Sequence logo representing sequence alignments of selected class II lantibiotic precursor peptides (for alignments, see Supplementary Fig. 3 online). The probability of each amino acid is depicted by the height of the letter and is scaled (width of the letter) according to how many sequences contributed to that position (i.e. narrow letters were generated from a smaller number of sequences than wider letters). Blue line above the sequence logo indicates the leader peptide, the ELXXBX motif is boxed in grey, and the double Gly motif is boxed in green.

Figure 3. Posttranslational modifications in microcin and cytolysin biosynthesis

a. Structures of microcin B17 and J25. b. Sequence logo representing sequence alignments of selected cytolysin precursor peptides (for alignments, see Supplementary Fig. 6 online). See the legend to Fig. 2b for further information about the logo format. The blue line above the sequence logo indicates the leader peptide, the FXXXB motif is boxed in grey, the TQV motif is boxed in violet, and the double Gly motif is boxed in green.

Figure 4. Proposed biosynthesis of patellamides C and A

The precursor peptide (PatE) consists of an N-terminal leader peptide and two core peptide cassettes, each sandwiched between two recognition sequences (green/violet). It is hypothesized that PatD is responsible for heterocyclization and PatG may be responsible for oxidation of the heterocycles. PatA sequentially removes cassette I and II from the leader peptide and the N-terminal recognition sequences (violet) and PatG removes the remaining recognition sequences (green) and catalyzes cyclization. PatF is essential for patellamide biosynthesis, but its precise role has not been identified.

Figure 5. Biosynthesis of thiostrepton

TsrH is ribosomally synthesized as a precursor peptide consisting of an N-terminal leader and C-terminal core peptide. Heterocyclization and dehydration of hydroxyl-amino acids results in a conformationally constrained core peptide, which is further tailored to include a central dehydropiperidine ring, a quinaldic acid moiety, and oxidative modifications. Although the transformations are shown in a particular order, the actual sequence of the modifications is not known. Alphabetical gene nomenclature is used as in .

Figure 6. Posttranslational modifications in conopeptides

a. Structures of the PTMs found in conopeptides. b. Sequence logo representing sequence alignments of selected contryphan precursor peptides (for alignments, see Supplementary Fig. 12 online). See the legend to Fig. 2b for further information about the logo format. The blue line above the sequence logo indicates the leader peptide, the green line above the sequence logo indicates the signal peptide, and the hypervariable core peptide region is boxed in yellow.