Mechanistic Probes for the Epimerization Domain of Nonribosomal Peptide Synthetases

Abstract

Nonribosomal peptide synthetases (NRPSs) are responsible for the synthesis of a variety of bioactive natural products with clinical and economic significance. Interestingly, these large multimodular enzyme machineries incorporate nonproteinogenic d-amino acids through the use of auxiliary epimerization domains, converting l-amino acids into d-amino acids that impart into the resulting natural products unique bioactivity and resistance to proteases. Due to the large and complex nature of NRPSs, several questions remain unanswered about the mechanism of the catalytic domain reactions. We have investigated the use of mechanism-based crosslinkers to probe the mechanism of an epimerization domain in gramicidin S biosynthesis. In addition, MD simulations were performed, showcasing the possible roles of catalytic residues within the epimerization domain.

Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism

“…Previously, pK a calculations indicated that the catalytic histidine residue was in a protonated state, suggesting that glutamate would act as a base in the epimerization mechanism. 14,18 This correlates with our data that in the wildtype TycA crosslinking, band A indicates 2.6 times more crosslinking than band B. For the H743A mutant crosslinking, where the catalytic residue E882 acts as a nucleophile, band A only shows 73% crosslinking compared to that of the wild-type crosslinking band A.…”

“…To further understand the roles of these active site residues, we decided to perform a diketopiperazine (DKP) assay, an in vitro condensation assay that allows us to indirectly study the activity of the E domain. 18,27 This assay allows us to quantify the dipeptide formed between the initiation module (TycA) and the subsequent downstream module TycB 1 (C-A-PCP Pro ), which is responsible for the selective uptake of epimerized D-Phe substrate in its condensation reaction. First, the A domains of TycA and TycB 1 catalyze the adenylation of L-Phe and L-Pro using ATP, and the activated amino acids are loaded onto their respective PCPs producing L-Phe-TycA and L-Pro-TycB 1 .…”

Thirtieth Anniversary of the Discovery of Laxaphycins. Intriguing Peptides Keeping a Part of Their Mystery

“…During reprotonation, an L/D mixture is obtained, but due to a strong specificity, the following C domain recognizes only the d -amino acids [ 91 ]. Different work is underway to shed light on the mechanism of action of these enzymes [ 92 ].…”

Bioactive Peptides Produced by Cyanobacteria of the Genus Nostoc: A Review

“…Similarly to NRPS, the synthesized PKs are modified by tailoring enzymes, such as ketoreductase, dehydratase, enoylreductase, or methyl transferase. The modifications often lead to increase in the bioactivity and the resistance of NRPs and PKs to enzymatic lysis [45]. They also generate large structural diversity within one class of compounds.…”