Substrate Specificity of the Lanthipeptide Peptidase ElxP and the Oxidoreductase ElxO

Abstract

The final step in lanthipeptide biosynthesis involves the proteolytic removal of an N-terminal leader peptide. In the class I lanthipeptide epilancin 15X, this step is performed by the subtilisin-like serine peptidase ElxP. Bioinformatic, kinetic, and mass spectrometric analysis revealed that ElxP recognizes the stretch of amino acids DLNPQS located near the proteolytic cleavage site of its substrate, ElxA. When the ElxP recognition motif was inserted into the noncognate lanthipeptide precursor NisA, ElxP was able to proteolytically remove the leader peptide from NisA. Proteolytic removal of the leader peptide by ElxP during the biosynthesis of epilancin 15X exposes an N-terminal dehydroalanine on the core peptide of ElxA that hydrolyzes to a pyruvyl group. The short-chain dehydrogenase ElxO reduces the pyruvyl group to a lactyl moiety in the final step of epilancin 15X maturation. Using synthetic peptides, we also investigated the substrate specificity of ElxO and determined the 1.85 Å resolution X-ray crystal structure of the enzyme.

Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes

“…278 As with other members of the SDR family, the architecture of ElxO is built around a Rossmann-fold dinucleotide-binding motif (Figure 17A). The NADP(H) cofactor binds in an extended conformation.…”

New Insights into the Biosynthetic Logic of Ribosomally Synthesized and Post-translationally Modified Peptide Natural Products

“…In addition, it has become apparent that different RiPP biosynthetic enzymes within the same pathway may recognize different recognition motifs within the same leader peptide (Goto et al, 2014, Ortega et al, 2014, Sardar et al, 2015b, Yang and van der Donk, 2013). Together, this knowledge may allow the use of leader peptide sequences as signature motifs to guide the installation of PTMs into non-cognate peptides.…”

Structure and mechanism of lanthipeptide biosynthetic enzymes

“…Indeed, mutations at residues Arg-1 and Ala-4 in the NisA substrate compromised substrate cleavage [53]. More recent work on the protease from the epilancin 15x biosynthetic cluster (ElxP) further subdivides the recognition elements into two clades: the (Ala/Gly-5)-(Ala-4)-x-x-(Arg-1) motif found in the C-terminus of the NisA leader sequence and related peptides (NisA class), and an (Asp/Glu-5)-(Leu/Val-4)-x-x-Gln-1 motif found in the epilancin 15x leader (ElxA) and related peptides (ElxA class) [54]. Sequence-swapping experiments demonstrated that protease specificity, within a respective clade, is governed by these five-residue motifs in the leader sequence.…”

“…More recently, the structure of the short-chain dehydrogenase ElxO involved in the maturation of epilancin 15X was solved (PDB: 4QEC) [54]. During the biosynthesis of this lanthipeptide, removal of the leader sequence exposes an amino-terminal Dha that hydrolyzes to a pyruvyl group.…”

“…The ElxO dehydrogenase then catalyzes the reduction of the ketone to generate an amino-terminal lactyl group in an NADP(H)-dependent manner. Mutational analysis identified several active site residues that function in catalysis, including Tyr152 that acts as the general acid and base, Lys156 that participates as an electrostatic catalyst, and Ser139 that polarizes the substrate peptide carbonyl [54]. Although a co-crystal structure with a substrate peptide is not available, a solvent-exposed surface diagram reveals an extensive groove along the active site trajectory that could serve as the peptide-binding site (Figure 4C).…”