The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain

Abstract

Peptide macrocycles are found in many biologically active natural products. Their versatility, resistance to proteolysis and ability to traverse membranes has made them desirable molecules. Although technologies exist to synthesize such compounds, the full extent of diversity found among natural macrocycles has yet to be achieved synthetically. Cyanobactins are ribosomal peptide macrocycles encompassing an extraordinarily diverse range of ring sizes, amino acids and chemical modifications. We report the structure, biochemical characterization and initial engineering of the PatG macrocyclase domain of Prochloron sp. from the patellamide pathway that catalyzes the macrocyclization of linear peptides. The enzyme contains insertions in the subtilisin fold to allow it to recognize a three-residue signature, bind substrate in a preorganized and unusual conformation, shield an acyl-enzyme intermediate from water and catalyze peptide bond formation. The ability to macrocyclize a broad range of nonactivated substrates has wide biotechnology applications.

Figure 1

Macrocyclization of patellamides. (a) The PatE prepropeptide consists of an N-terminal leader sequence followed by two eight-residue cassettes with the C-terminal macrocyclase recognition signal AYDG. X indicates any amino acid. The macrocyclization domain of PatG catalyzes the formation (dashed lines) of two cyclic peptides per prepropeptide. (b) PatGmac requires a heterocycle or proline (denoted Z) at the P1 position and the AYDG motif at the P1′–P4′ sites. An additional glutamate is often found at P5′ but is not required. (c) The test substrate used in this study can either give a linear peptide (curved line) with a mass of 716.375 Da or a macrocycle (octagon), which is 18 Da lighter.

Figure 2

Structure of PatGmac. (a) PatGmac has a subtilisin-like core (cyan) and the conventional catalytic triad (yellow sticks). PatGmac has an insertion (magenta) that extends from β2 as a loop, then forms a helix-loop-helix motif and creates an N-terminal extension of α4, the helix that harbors His618. The insertion is found in other macrocyclases but is not conserved in length or sequence. (b) Difference electron density (F_o – F_c contoured at 3σ with phases calculated from a model which was refined with no peptide present) of the PIPFPAYDG substrate mimic bound to PatGmac H618A; three N-terminal residues (VPA) of the substrate mimic are disordered. (c) Interactions between the substrate mimic and PatGmac H618A. The proline (at position P1) of the substrate adopts a cis peptide conformation that results in the substrate pointing away from the protein. The side chains of Met660, Phe684 and Arg686 would prevent the binding of substrates that adopt an extended conformation. Lys598 and possibly Lys594 form salt bridges (dashed lines) with the P3′ residue (aspartate), whereas the P2′ tyrosine forms a hydrogen bond (dashed line) with His746 and interacts with Phe747 through π-stacking. (d) The active site where the acyl-enzyme intermediate would be formed is shielded from solvent by the macrocyclization insertion and the AYDG peptide (dark green).

Figure 3

Biochemical characterization of PatGmac and PatGmac mutants. (a–c) LC-ESI MS of macrocyclization reactions with PatGmac wild type (a), PatGmacΔ2 (b) and PatGmac K594D (c). Macrocyclized and linear products are indicated with octagons and curved lines, respectively. The error between observed and calculated mass is shown below the [M+H]⁺ and [M+Na]⁺ species. (d) Evidence for a stable acyl-enzyme intermediate between PatGmac and substrate.

Figure 4

Proposed mechanism for macrocyclization. (a) Model of the acyl-enzyme intermediate with AYDG remaining bound at the active site. (b) The acyl-enzyme intermediate is in equilibrium with the substrate. In PatGmac the N terminus of the substrate enters the active site, displacing AYDG and leading to macrocyclization. Mutations that disrupt binding of AYDG lead to linear product, as the substrate is hydrolyzed by water. The role of the histidine in deprotonating the incoming N terminus is speculative.