Journal article
Biosynthesis of a Novel Glutamate Racemase Containing a Site-Specific 7-Hydroxycoumarin Amino Acid: Enzyme–Ligand Promiscuity Revealed at the Atomistic Level
ACS central science, Vol.1(7), pp.364-373
10/28/2015
DOI: 10.1021/acscentsci.5b00211
PMCID: PMC4626791
PMID: 26539562
Abstract
Glutamate
racemase (GR) catalyzes the cofactor independent stereoinversion
of
l
- to
d
-glutamate for biosynthesis of bacterial
cell walls. Because of its essential nature, this enzyme is under
intense scrutiny as a drug target for the design of novel antimicrobial
agents. However, the flexibility of the enzyme has made inhibitor
design challenging. Previous steered molecular dynamics (MD), docking,
and experimental studies have suggested that the enzyme forms highly
varied complexes with different competitive inhibitor scaffolds. The
current study employs a mutant orthogonal tRNA/aminoacyl-tRNA synthetase
pair to genetically encode a non-natural fluorescent amino acid,
l
-(7-hydroxycoumarin-4-yl) ethylglycine (7HC), into a region
(Tyr53) remote from the active site (previously identified by MD studies
as undergoing ligand-associated changes) to generate an active mutant
enzyme (GR
Y53/7HC
). The GR
Y53/7HC
enzyme is
an active racemase, which permitted us to examine the nature of these
idiosyncratic ligand-associated phenomena. One type of competitive
inhibitor resulted in a dose-dependent quenching of the fluorescence
of GR
Y53/7HC
, while another type of competitive inhibitor
resulted in a dose-dependent increase in fluorescence of GR
Y53/7HC
. In order to investigate the environmental changes of the 7HC ring
system that are distinctly associated with each of the GR
Y53/7HC
–ligand complexes, and thus the source of the disparate quenching
phenomena, a parallel computational study is described, which includes
essential dynamics, ensemble docking and MD simulations of the relevant
GR
Y53/7HC
–ligand complexes. The changes in the solvent
exposure of the 7HC ring system due to ligand-associated GR changes
are consistent with the experimentally observed quenching phenomena.
This study describes an approach for rationally predicting global
protein allostery resulting from enzyme ligation to distinctive inhibitor
scaffolds. The implications for fragment-based drug discovery and
high throughput screening are discussed.
A mutant glutamate racemase enzyme possessing
a genetically
encoded non-natural fluorescent amino acid is developed and studied
as a reporter of global enzyme changes occurring upon ligand binding.
Details
- Title: Subtitle
- Biosynthesis of a Novel Glutamate Racemase Containing a Site-Specific 7-Hydroxycoumarin Amino Acid: Enzyme–Ligand Promiscuity Revealed at the Atomistic Level
- Creators
- Sondra F Dean - Division of Medicinal and Natural Products Chemistry, College of Pharmacy, andKatie L Whalen - Department of BiochemistryM. Ashley Spies - Division of Medicinal and Natural Products Chemistry, College of Pharmacy, and
- Resource Type
- Journal article
- Publication Details
- ACS central science, Vol.1(7), pp.364-373
- DOI
- 10.1021/acscentsci.5b00211
- PMID
- 26539562
- PMCID
- PMC4626791
- NLM abbreviation
- ACS Cent Sci
- ISSN
- 2374-7943
- eISSN
- 2374-7951
- Publisher
- American Chemical Society
- Grant note
- DOI: 10.13039/100000057, name: National Institute of General Medical Sciences, award: R01-GM097373
- Language
- English
- Date published
- 10/28/2015
- Academic Unit
- Pharmaceutical Sciences and Experimental Therapeutics; Biochemistry and Molecular Biology; Medicinal and Natural Products Chemistry
- Record Identifier
- 9984025258802771
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