Probing the Compatibility of an Enzyme‐Linked Immunosorbent Assay toward the Reprogramming of Nonribosomal Peptide Synthetase Adenylation Domains

Abstract

An important challenge in natural product biosynthesis is the biosynthetic design and production of artificial peptides. One of the most promising strategies is reprogramming adenylation (A) domains to expand the substrate repertoire of nonribosomal peptide synthetases (NRPSs). Therefore, the precise detection of subtle structural changes in the substrate binding pockets of A domains might accelerate their reprogramming. Here we show that an enzyme-linked immunosorbent assay (ELISA) using a combination of small-molecule probes can detect the effects of substrate binding pocket residue substitutions in A-domains. When coupled with a set of aryl acid A-domain variants (total of nine variants), the ELISA can analyze the subtle differences in their active-site architectures. Furthermore, the ELISA-based screening was able to identify the variants with substrate binding pockets that accepted a non-cognate substrate from an original pool of 45. These studies demonstrate that ELISA is a reliable platform for providing insights into the active-site properties of A-domains and can be applied for the reprogramming of NRPS A-domains.

Activity, Binding, and Modeling Studies of a Reprogrammed Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket

“…10) More recently, we confirmed that the N235G variant displays enzymatic activity toward 2-ABA by a proof-of-concept study of an enzyme-linked immunosorbent assay system (ELISA) for the A-domains in NRPSs. 11) The binding profiles only provide information on substrate candidates of wild-type EntE and the N235G variant, but unable to predict adenylation activity. However, substrate recognition is an important event for enzyme catalysis.…”

“…9) Recently, we facilitated the precise understanding of NRPS A-domain codes of aryl acids by gradually grafting the NRPS codes of salicylic acid-activating A-domains into the substrate binding pocket of EntE. 10) More recently, we confirmed that the N235G variant displays enzymatic activity toward 2-ABA by a proof-of-concept study of an enzyme-linked immunosorbent assay system (ELISA) for the A-domains in NRPSs. 11) The binding profiles only provide information on substrate candidates of wild-type EntE and the N235G variant, but unable to predict adenylation activity.…”

Structural, biochemical and bioinformatic analyses of nonribosomal peptide synthetase adenylation domains

“…Some strategies that will not be discussed in detail here include the body of work pioneered by the Ishikawa and Kakeya labs in the realm of chemical proteomics and activity-based protein proling. 110,111 Building on work that designed and synthesized clickable probes 82 and inhibitors of A domains, [55][56][57] Ishikawa and coworkers have established methods that can detect and interrogate endogenous NRPSs using ELISA [112][113][114] and in vivo techniques. 115,116 This has provided valuable insight into chemical moieties required for binding and remodeling of active site architecture, while avoiding the labor-intensive and problematic enzyme purications that many other workows require.…”