Interrupted adenylation domains: unique bifunctional enzymes involved in nonribosomal peptide biosynthesis

Abstract

Nonribosomal peptides (NRPs) account for a large portion of drugs and drug leads currently available in the pharmaceutical industry. They are one of two main families of natural products biosynthesized on megaenzyme assembly-lines composed of multiple modules that are, in general, each comprised of three core domains and on occasion of accompanying auxiliary domains. The core adenylation (A) domains are known to delineate the identity of the specific chemical components to be incorporated into the growing NRPs. Previously believed to be inactive, A domains interrupted by auxiliary enzymes have recently been proven to be active and capable of performing two distinct chemical reactions. This highlight summarizes current knowledge on A domains and presents the various interrupted A domains found in a number of nonribosomal peptide synthetase (NRPS) assembly-lines, their predicted or proven dual functions, and their potential for manipulation and engineering for chemoenzymatic synthesis of new pharmaceutical agents with increased potency.

Structural insight into the necessary conformational changes of modular nonribosomal peptide synthetases

“…Finally, termination modules contain an additional thioesterase or reductase domain that catalyzes the final release of the product through hydrolysis, reduction, or cyclization. Other domains have been identified within NRPS modules to catalyze N-methylation, epimerization, cyclization, oxidation/reduction, or additional chemical modifications to the amino acid or peptide intermediates [10,11]. …”

Expanding Substrate Promiscuity by Engineering a Novel Adenylating‐Methylating NRPS Bifunctional Enzyme

“…These conserved core sequences play important roles in substrate binding and catalysis . Some unique A domains have been found to be interrupted between two core sequences by portions of auxiliary domains such as M, Ox, MOx, and ketoreductase (KR) . Although interruptions are most commonly found between a8 and a9, there is one example of an A domain interrupted by a portion of an M domain between a2 and a3: TioN from the thiocoraline biosynthetic pathway .…”

“…[3] Some unique Adomains have been found to be interrupted between two core sequences by portions of auxiliary domains such as M, Ox, MOx, and ketoreductase (KR). [4] Although interruptions are most commonly found between a8 and a9, there is one example of an Ad omain interrupted by ap ortion of an M domain between a2 anda 3: TioN from the thiocoraline biosyntheticp athway. [5] The CesA and CesB domains and the Vlm1 and Vlm2Adomains are interrupted between a8 and a9 by aK Ri nc ereulide and valinomycin biosynthetic pathways, respectively,a nd have been shown to be capable of both adenylation and ketoreduction of their a-keto acid cognates ubstrates tethered to Td omains.…”

“…As expected, we did not observe any adenylating activity with the negative control, KtzJ alone. In order to confirm that Ni II -NTAa ffinity chromatography afforded proteins of sufficient purity,w ea lso performed an ATP-[ 32 P]PP i exchange assay with our KtzH(A 4a DMA 4b T 4 )p rotein that we further purified ( Figure S3), with l-Ser as the substrate. Only protein in FPLC fractions1 0a nd 11 activated l-Ser,s imilarly to the protein purified solely by Ni II -NTAa ffinity chromatography.…”

“…We are currently investigating the potentialo fc reating novel bifunctional and trifunctional enzymesw ith adenylating and other activities from monofunctional Adomains from variousNRPS systems. (Table S1), PCR strategies used for preparation of the ktzH(A 4a DMA 4b T 4 ) and ktzH(A 4a M N A 4b T 4 ) (Table S2)…”