Insights into the Mechanism of the Cyanobactin Heterocyclase Enzyme.

Ying Ge,Ona K Miller,Catherine H Botting,James H Naismith,Ulrich Schwarz-Linek,Clarissa Melo Czekster

doi:10.1021/acs.biochem.9b00084

Abstract

Cyanobactin heterocyclases share the same catalytic domain (YcaO) as heterocyclases/cyclodehydratases from other ribosomal peptide (RiPPs) biosynthetic pathways. These enzymes process multiple residues (Cys/Thr/Ser) within the same substrate. The processing of cysteine residues proceeds with a known order. We show the order of reaction for threonines is different and depends in part on a leader peptide within the substrate. In contrast to other YcaO domains, which have been reported to exclusively break down ATP into ADP and inorganic phosphate, cyanobactin heterocyclases have been observed to produce AMP and inorganic pyrophosphate during catalysis. We dissect the nucleotide profiles associated with heterocyclization and propose a unifying mechanism, where the γ-phosphate of ATP is transferred in a kinase mechanism to the substrate to yield a phosphorylated intermediate common to all YcaO domains. In cyanobactin heterocyclases, this phosphorylated intermediate, in a proportion of turnovers, reacts with ADP to yield AMP and pyrophosphate.

Highlights

Ribosomal peptide natural products, known as RiPPs, are an intriguing class of genetically encoded post-translationally modified molecules produced by bacteria, plants, and fungi.[1]
The reaction was analyzed by MALDI-MS without (Figure 1A) and with (Figure 1A,B) the sample being treated with iodoacetamide (IAA), an alkylating agent that covalently modifies cysteine residues resulting in the addition of 57 Da mass per free cysteine (Figure 1A,B)
The E. coli YcaO protein was observed to produce AMP and PPi in the absence of a substrate, but since the substrate is unknown, the relative rates cannot be estimated for YcaO.[18]

Summary

Introduction

Known as RiPPs, are an intriguing class of genetically encoded post-translationally modified molecules produced by bacteria, plants, and fungi.[1]. The five-membered heterocyclic rings (azol(in)es) are found in a wide range of RiPPs that include the linear azol(in)econtaining peptides (known as LAPS), cyanobactins, thiopeptides, and bottromycins.[1] The ring results from the formation of a bond between the amino acid side chain oxygen or sulfur atom (from cysteine, serine, or threonine) and the preceding amide bond.[13] ATP and Mg2+ are required for heterocyclization enzyme activity, and all heterocyclase enzymes share the same catalytic unit, the YcaO domain (named after the E. coli homologue).[14] The YcaO domain works in conjunction with a substrate recognition unit, known as the RiPP recognition element (RRE),[15] which can either be fused to the YcaO domain or occur as a separate protein. An enzyme operating via two different mechanisms for the same substrate would seem highly unlikely; rather, the observations could point to something important that has been overlooked

Methods

Results

Conclusion