The Cyanobactin Heterocyclase Enzyme: A Processive Adenylase That Operates with a Defined Order of Reaction**

Heterocyclic rings are a recurring and iconic motif in organic chemistry. The introduction of five-membered rings into protein backbones is synthetically challenging, but many peptide-based biologically active natural products contain such Ser-, Thr-, and Cys-derived heterocycles and therefore a facile route to their introduction is highly desirable. More generally thiazolines and oxazolines, as well as their oxidized forms (thiazoles and oxazoles), are found in a variety of approved drugs, drug leads, and toxins. The cyanobacterium Prochloron spp. produces multiple macrocyclic cyanobactins, which are known as the trunkamides (seven or eight residues) and the patellamides (eight residues). These natural products originate from two different ribosomal precursor peptides (denoted TruE and PatE), and each ribosomal peptide contains multiple distinct core peptides. In TruE (and PatE), each of the core peptides is flanked at the N-terminus by a conserved five-residue protease signature and at the C-terminus by a conserved macrocyclization signature (A/SYDG; Scheme 1). It is the core peptides that go on to become the different natural products. In trunkamides and patellamides (as well as other cyanobactins) these products possess multiple heterocyclic cysteine, and/or serine and threonine amino acids. All cyanobactin ribosomal precursor peptides possess (along with the core peptides and their flanking regions) a conserved thirtyto forty-residue N-terminus (Scheme 1). This Nterminal leader, which is discarded during processing is thought to be essential for heterocyclization. Heterocyclization of the multiple cysteine residues in TruE is carried out by the single enzyme, TruD, which by definition is processive. The amino acid sequence of the core peptide is variable (Supporting Information, Figure S1) and thus TruD must be in part insensitive to the immediate sequence context of the target cysteine (Scheme 1). Further, the positions of the target cysteines in TruE relative to the leader vary, both within and between core peptides. This flexibility suggests that TruD could be valuable for synthetic chemists. The corresponding enzyme in the patellamide pathway is denoted PatD. TruD and PatD process cysteine residues in the other enzymes precursor peptide substrate equally well; TruD processes serine and threonine residues less well than PatD. The interchangeability of enzymes mirrors the high degree of sequence identity in the enzymes (Figure S2) and substrate Nterminal leader (Figure S1). The heterocyclization of cysteine to form thiazoline as part of the microcins (thiazole/oxazole-modified microcins, or TOMMs) has recently been studied and the responsible ATP Mg dependent heterocyclase BalhD identified. BalhD is homologous to the C-terminal 400 residues of TruD, whilst a second protein BalhC matches the N-terminal 300 residues (Figure S2). When both BalhD and BalhC are present heterocyclization of the microcin substrate is accelerated and only when both proteins are present can phosphate release be robustly measured. Pyrophosphate production by BalhD was ruled out and BalhD was proposed to operate by Scheme 1. Heterocyclization reaction catalyzed in the cyanobactins. TruD efficiently processes cysteines and selenocysteine. TruD operates on core peptides of seven or eight residues; a single precursor peptide can contain up to four core peptides each with different sequences. The immediate sequence context and position (relative to the leader) of the target cysteine is variable.