Contribution of Water and Energetics of Ligand Binding in the Catalytic Mechanism of R67 Dihydrofolate Reductase

R67 dihydrofolate reductase (DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate (DHF) to produce tetrahydrofolate (THF). The enzyme is a homotetramer and its 222 symmetry allows for binding of both ligands to a single active site pore. A productive ternary complex is formed by the binding of one molecule of DHF and NADPH and inter-ligand cooperativity has been suggested to be essential for binding and catalysis. To gain further insight into the thermodynamics involved in the ground state and the transition state, temperature dependent studies on DHF binding and catalysis were performed. It was observed that binding of both NADPH and DHF is enthalpy driven. From van’t Hoff plots, the change in enthalpy, entropy and free energy for NADPH binding to R67 DHFR in the ground state were determined. Similarly, the thermodynamics of DHF binding to the R67 DHFR-NADPH complex in the ground state were determined. Arrhenius plots were also employed to study the energetics of the transition state. A comparison of TdeltaS values (for DHF binding to R67 DHFR-NADPH complex) in both ground state and transition state indicates that TdeltaS is more negative in the transition state (–11.3 kcal/mol) as compared to the ground state (–5.4 kcal/mol). This indicates a reorientation of the substrate in the transition state. The role of water in DHF and NADPH binding to R67 DHFR was also investigated. For this, the effect of osmotic pressure on the Ka /Km of ligand binding, as well as the kcat of the reaction was studied. It was observed that the kcat of the reaction was not significantly affected, while the binding of ligands was affected with increasing osmolality. Specifically, binding of NADPH tightened as osmolality increased, while binding of DHF weakened with increasing osmolality, suggesting release of water upon NADPH binding and an uptake of water on DHF binding. Results from in vivo experiments on E.coli cells containing wild type and mutant clones of R67 DHFR were also consistent with in vitro experiments, suggesting that water is involved in ligand binding to R67 DHFR

tRNAs as Antibiotic Targets

Transfer RNAs (tRNAs) are central players in the protein translation machinery and as such are prominent targets for a large number of natural and synthetic antibiotics. This review focuses on the role of tRNAs in bacterial antibiosis. We will discuss examples of antibiotics that target multiple stages in tRNA biology from tRNA biogenesis and modification, mature tRNAs, aminoacylation of tRNA as well as prevention of proper tRNA function by small molecules binding to the ribosome. Finally, the role of deacylated tRNAs in the bacterial “stringent response” mechanism that can lead to bacteria displaying antibiotic persistence phenotypes will be discussed

Structural characterization of antibiotic self-immunity tRNA synthetase in plant tumour biocontrol agent

Antibiotic-producing microbes evolved self-resistance mechanisms to avoid suicide. The biocontrol Agrobacterium radiobacter K84 secretes the Trojan Horse antibiotic agrocin 84 that is selectively transported into the plant pathogen A. tumefaciens and processed into the toxin TM84. We previously showed that TM84 employs a unique tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase (LeuRS), while the TM84-producer prevents self-poisoning by expressing a resistant LeuRS AgnB2. We now identify a mechanism by which the antibiotic-producing microbe resists its own toxin. Using a combination of structural, biochemical and biophysical approaches, we show that AgnB2 evolved structural changes so as to resist the antibiotic by eliminating the tRNA-dependence of TM84 binding. Mutagenesis of key resistance determinants results in mutants adopting an antibiotic-sensitive phenotype. This study illuminates the evolution of resistance in self-immunity genes and provides mechanistic insights into a fascinating tRNA-dependent antibiotic with applications for the development of anti-infectives and the prevention of biocontrol emasculation

Emerging Approaches for Fluorescence-Based Newborn Screening of Mucopolysaccharidoses

Interest in newborn screening for mucopolysaccharidoses (MPS) is growing, due in part to ongoing efforts to develop new therapies for these disorders and new screening assays to identify increased risk for the individual MPSs on the basis of deficiency in the cognate enzyme. Existing tests for MPSs utilize either fluorescence or mass spectrometry detection methods to measure biomarkers of disease (e.g., enzyme function or glycosaminoglycans) using either urine or dried blood spot (DBS) samples. There are currently two approaches to fluorescence-based enzyme function assays from DBS: (1) manual reaction mixing, incubation, and termination followed by detection on a microtiter plate reader; and (2) miniaturized automation of these same assay steps using digital microfluidics technology. This article describes the origins of laboratory assays for enzyme activity measurement, the maturation and clinical application of fluorescent enzyme assays for MPS newborn screening, and considerations for future expansion of the technology

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase

9 pags, 5 figs, 2 tabsLeucyl-tRNA synthetases (LeuRSs) have an essential role in translation and are promising targets for antibiotic development. Agrocin 84 is a LeuRS inhibitor produced by the biocontrol agent Agrobacterium radiobacter K84 that targets pathogenic strains of A. tumefaciens, the causative agent of plant tumours. Agrocin 84 acts as a molecular Trojan horse and is processed inside the pathogen into a toxic moiety (TM84). Here we show using crystal structure, thermodynamic and kinetic analyses, that this natural antibiotic employs a unique and previously undescribed mechanism to inhibit LeuRS. TM84 requires tRNA Leu for tight binding to the LeuRS synthetic active site, unlike any previously reported inhibitors. TM84 traps the enzyme-tRNA complex in a novel 'aminoacylation-like' conformation, forming novel interactions with the KMSKS loop and the tRNA 3′-end. Our findings reveal an intriguing tRNA-dependent inhibition mechanism that may confer a distinct evolutionary advantage in vivo and inform future rational antibiotic design. © 2013 Macmillan Publishers Limited. All rights reserved

Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase

Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the <i>p</i>-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ∼35% were constructed. Only minor effects on <i>k</i>_cat were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)­propyl]­carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the <i>p</i>-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The <i>K</i>_i for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis