title: 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.
abstract: 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'-O-N-(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 Escherichia coli 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 kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km 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.
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University , Sakyo, Kyoto 606-8501, Japan.
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Ishikawa
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University , Sakyo, Kyoto 606-8501, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Miyamoto
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University , Sakyo, Kyoto 606-8501, Japan.
authors.forename: Sho
authors.initials: S
authors.lastname: Konno
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University , Sakyo, Kyoto 606-8501, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Kasai
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University , Sakyo, Kyoto 606-8501, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kakeya
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry*; D001665:Binding Sites; D020134:Catalytic Domain; D004797:Enzyme-Linked Immunosorbent Assay; D004800:Enzymes, Immobilized / Q000737:chemistry; D015336:Molecular Probe Techniques; D015394:Molecular Structure; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D004800:Enzymes, Immobilized; D000249:Adenosine Monophosphate; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase