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

摘要

A significant, gap exists between protein engineering and enzymes used for the biosynthesis of natural, products, largely because 1;, there is a paucity of strategies:that rapidly detect. active site phenotypes the enzymes with desired activities. Herein, we describe a proof-oft : concept study of an enzyme-linked immunosorbe4-asaay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetase (NRPSs)' using a,combination of active site-directed probes coupled a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality, that, immobilizes probe xriOlectles onto a Streptavidin-coated solid Support. The recombinant NRPSs have a C-terrinnal His tag motif that is targeted by an anti-6xHis 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 domains in the crude cell-free homogenates from the Escherichia,colt 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-dihydroxybenxoic 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 k(cat)/K-m value with the noncognate substrate Sal and a corresponding 48 fold decrease in the k(cat)/K-m 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.