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.