Articles pubs.acs.org/acschemicalbiology
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 Fumihiro Ishikawa,* Kengo Miyamoto, Sho Konno, Shota Kasai, and Hideaki Kakeya* 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 S Supporting Information *
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-ofconcept 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.
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aminoacyl-S-T (Figure 1a). The simple biochemical logic of the A domains has made them attractive targets for metabolic engineering,5 combinatorial biosynthesis,6 and directed evolution7−9 for the production of new small molecule therapeutics. However, the rational design of the active sites of A domains with programmed substrate specificities is challenging. This is largely because of the lack of methods to guide the manipulation of the active sites of A domains. Traditional methods to assess the enzymatic activities of the A domains of NRPSs rely on radioactive methods such as ATP[32P]pyrophosphate (PPi) exchange.10 These methods use an assay that has several laborious, complicated, and timeconsuming steps involving handling radioactivity. In addition,
onribosomal peptide synthetases (NRPSs) are large, highly versatile multifunctional enzymes that are responsible for the biosynthesis of peptide-based natural products known as nonribosomal peptides (NRPs), which are structurally diverse and display a broad range of important biologically activities.1 The adenylation (A) domain selectively incorporates cognate amino acids into NRPs from a much larger monomer pool, including all 20 proteinogenic amino acids, as well as a number of nonproteinogenic amino acids, aryl acids, fatty acids, and hydroxy acid building blocks.2−4 The A domain initially recognizes a cognate amino acid and converts it to the corresponding aminoacyl adenylate intermediate at the expense of Mg2+ and a molecule of ATP with the release of PPi (Figure 1a). The adenylated substrate subsequently undergoes nucleophilic attack by the terminal thiol group of the 4′phosphopantetheine (PPant) arm of a downstream thiolation (T) domain, leading to the formation of a thioester bound © XXXX American Chemical Society
Received: July 28, 2015 Accepted: September 26, 2015
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DOI: 10.1021/acschembio.5b00595 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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ACS Chemical Biology
Figure 1. (a) Adenylation reaction in NRPS. (b) Structures of active site-directed probes and cognate competitors of the probes for A domains in NRPSs described in this study.
the ATP-[32P]PPi exchange assay requires relatively large amounts of [32P]PPi, and the presence of high background signals complicates data analysis. Modern variants of the ATP[32P]PPi exchange assay have recently been developed for applications in high-throughput programs and kinetics measurement.11 However, not all facilities are equipped to manipulate radioactive compounds in a high-throughput program. A second strategy used to determine the activity of the A domains of NRPSs is the loading of radiolabeled amino acid substrates onto the proteins for autoradiographic analysis following SDS-PAGE gel electrophoresis.12,13 While SDSPAGE gel analysis is the most commonly practiced technique, it is not useful if the radiolabeled amino acid substrates are not commercially available. In addition, the labile thioester bonds that hold the radiolabeled amino acid substrates to the PPant arm of the T domains of NRPS proteins are readily hydrolyzable. Garneau-Tsodikova and co-workers have reported a convenient high-throughput nonradioactive technique (96- or 384-well formats) to assess the activity of the A domains of NRPSs.14 In this assay, a molybdate/malachite green reagent15 is used to assess the concentration of orthophosphate (Pi) produced after treatment of the inorganic pyrophosphate of the PPi released during aminoacyl-AMP formation catalyzed by the A domains. In addition, Aldrich and Wilson recently described a coupled continuous assay that uses hydroxylamine as a surrogate acceptor molecule, resulting in the formation of a hydroxamate.16 The released PPi is detected using a pyrophosphatase-purine nucleoside phosphorylase coupling system with the chromogenic substrate 7-methylthioguanosine (MesG).17 This continuous, nonradioactive hydroxamate-MesG assay enabled rapid measurement of the activity of A domains and the apparent inhibition constants for inhibitor−enzyme pairs. However, these techniques are limited to the analysis of purified proteins. New strategies are continually being developed for the analysis of nonribosomal and polyketide biosynthetic machineries. Rapid, highly
sensitive, and nonradioactive mass spectrometry (MS)-based techniques have been developed to investigate these enzyme families. Bachmann and co-workers have described a MS-based γ-18O4-ATP pyrophosphate exchange assay to measure Adomain specificity.18 This method has several practical advantages, including the use of stable isotopes, safe handling, and the speed of the MS-based assay, but it requires rapid exchange of PPi with the acyl adenylate. Dorrestein and coworkers have reported an elegant MS-based strategy, known as a PPant ejection assay, that enables the analysis of substrates, intermediates, and products attached to the PPant arm of the carrier proteins of nonribosomal and polyketide biosynthetic enzymes.19 Tandem MS methods are used to eject the PPant arm from the intact protein and peptide forming two ejection ions, which results in the assignment of the covalently tethered biosynthetic intermediate structures. The main advantage to this method is that the PPant ejection rapidly reduces large (>100 kDa) enzymes to
