10.1021/acschembio.5b00595.s001 Fumihiro Ishikawa Fumihiro Ishikawa Kengo Miyamoto Kengo Miyamoto Sho Konno Sho Konno Shota Kasai Shota Kasai Hideaki Kakeya Hideaki Kakeya Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains American Chemical Society 2015 nonribosomal peptide synthetases immobilizes probe molecules enzyme Adenylation Domain Functions antibody Nonribosomal Peptide Synthetases noncognate substrate Sal ELISA technique NRPS EntE laboratory ELISA technique DHB Escherichia coli expression systems 2015-12-18 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Accurate_Detection_of_Adenylation_Domain_Functions_in_Nonribosomal_Peptide_Synthetases_by_an_Enzyme_linked_Immunosorbent_Assay_System_Using_Active_Site_directed_Probes_for_Adenylation_Domains/2097052 A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5′-<i>O</i>-<i>N</i>-(aminoacyl)­sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the <i>Escherichia coli</i> expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe–protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> value with the noncognate substrate Sal and a corresponding 48-fold decrease in the <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique and adenylating enzymes together using a combination of active site-directed probes for the A domains in NRPSs should accelerate both the functional characterization and manipulation of the A domains in NRPSs.