Peptide Crosslinking by a Class of Plant Copper Enzymes

Abstract

BURP domain peptide cyclases, or BpCs (an abbreviation we recommend in this opinion), are an emerging class of copper enzymes which catalyze the oxidative macrocyclization of peptides in plants. A close examination of their novel protein fold, along with the unique dicopper active site that meticulously controls crosslinking within peptides, highlights how nature exploits intricate mechanistic strategies to achieve diverse functionalities. Here, we summarize recent discoveries regarding the sequence, structure, function, and proposed chemistry of BpCs. We also present plausible mechanistic ideas and recommend important structural considerations that could advance investigations and discussions surrounding their reactivity and underlying mechanisms.

Keywords: Copper enzymes; Dioxygen chemistry; Oxidative peptide macrocyclization; Plant development.

Figure 1.

The primary classification and function of BpC. (A) Primary structures of founding members of BURP domain family adapted from reference [4] along with our proposed classification of the key conserved features, i.e., the surface cysteine pair (CC-S), the pairs of cysteine-histidine motifs located on strands 6 and 7 of the β-sheet (CC-A and HH-A), and those on strands 9 and 10 of the semi-β-barrel (CC-B and HH-B). Note: Additional subfamilies have emerged over the recent years, including BURP-V, BURP-VI, and BURP-VII, that are not included here. (B) Fused and split BpCs based on the proximity of the precursor peptide and enzyme producing RiPPs or post-translationally modified (PTMed) proteins. (C) Subcellular localization of BpCs in plant cells. Bold indicates where most genes are found. (D) Structures of four examples of BpC-derived macrocyclic RiPPs.

Figure 2.

The structure and proposed mechanisms of BpCs. (A) The active site of AhyBURP showing the copper ligand environment with binding occurring at the Nε atom of both histidines, similar to that of Cu_M in PHM. The following BpC features are highlighted: CC-S in yellow, CC-A and HH-A in blue, and CC-B and HH-B in red. Displayed are: (top) single copper coordinated in chain A of PDB ID: 8SY3, (middle) water-bound copper coordination in chain B of PDB ID: 8SY3, and (bottom) active site of the AlphaFold 3 predicted protein with same sequence, where copper coordinating on HH-B, with the tryptophan of the core peptide within ~3 Å from the copper center. (B) AlphaFold 3 predicted “hinge” or “butterfly” like structure of a split BpC from C. arabica with a precursor peptide (in orange) highlighting the copper coordination on both sides of the active site. The redox-active aromatic amino acid tyrosine from the core peptide is in close proximity of the copper center, where the O₂ chemistry occurs. (C) Examples of copper/O₂ adducts (top) which can form at one or two copper centers and reaction mechanisms (bottom) showing the formation of cupryl radical in an uncoupled mechanism and formation of the closed active species, Cu(II)-(μ-O•)(μ-OH)-Cu(II), in a coupled mechanism. (D) The balanced reaction equation catalyzed by BpCs reducing O₂ to water by four electrons along with the oxidative coupling of the substrate. The two electrons and protons are initially delivered to the active site through the proposed cysteine-dithiol/disulfide electron relay.

Figure 3, Key Figure.

The key features of the BpC active site and the proposed chemistry involved. Key structural features of BpCs and our recommended assignments are presented. In the precursor and crosslinked peptides, Z represents any amino acid, while Y is a redox-active aromatic amino acid. The reaction at the active site illustrates the complete four-electron (4 ē) reduction of dioxygen to two water molecules (red arrow). Two electrons and two protons are provided by the precursor peptide, leading to the formation of a crosslinked peptide (green arrow). We propose that the additional two electrons and two protons are likely delivered to the dicopper active site via the CC-S and CC-A thiol/disulfide pairs (broken arrows).