Loop Interactions during Catalysis by Dihydrofolate Reductase fromMoritella profunda

Dihydrofolate reductase (DHFR) is often used as a model system to study the relation between protein dynamics and catalysis. We have studied a number of variants of the cold-adapted DHFR from Moritella profunda (MpDHFR), in which the catalytically important M20 and FG loops have been altered, and present a comparison with the corresponding variants of the wellstudied DHFR from Escherichia coli (EcDHFR). Mutations in the M20 loop do not affect the actual chemical step of transfer of hydride from reduced nicotinamide adenine dinucleotide phosphate to the substrate 7,8-dihydrofolate in the catalytic cycle in either enzyme; they affect the steady state turnover rate in EcDHFR but not in MpDHFR. Mutations in the FG loop also have different effects on catalysis by the two DHFRs. Despite the two enzymes most likely sharing a common catalytic cycle at pH 7, motions of these loops, known to be important for progression through the catalytic cycle in EcDHFR, appear not to play a significant role in MpDHFR

NMR Structures of Apo L. casei Dihydrofolate Reductase and Its Complexes with Trimethoprim and NADPH: Contributions to Positive Cooperative Binding from Ligand-Induced Refolding, Conformational Changes, and Interligand Hydrophobic Interactions

bS Supporting Information The enzyme dihydrofolate reductase (DHFR; 5,6,7,8-tetra-hydrofolate:NADPH oxidoreductase, EC 1.5.1.3) catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydro-folate (THF) using NADPH as coenzyme.1 Since THF and its metabolites are precursors of purine and pyrimidine bases, the normal functioning of this enzyme is essential for proliferating cells. This makes DHFR an excellent target for antifolate drugs such as methotrexate (anticancer), pyrimethamine (antimalarial), and trimethoprim (antibacterial). Such agents act by inhibiting the enzyme in parasitic or malignant cells.1,2 The cooperative binding of ligands to DHFR plays an important role not only in the enzyme catalytic cycle (negative cooperativity in THF/ NADPH binding)3 but also in enzyme inhibition (positive cooperativity in antifolate/NADPH binding).4 The effects of positive cooperative binding in controlling enzyme inhibition ar

Structure and Dynamics of the G121V Dihydrofolate Reductase Mutant: Lessons from a Transition-State Inhibitor Complex

It is well known that enzyme flexibility is critical for function. This is due to the observation that the rates of intramolecular enzyme motions are often matched to the rates of intermolecular events such as substrate binding and product release. Beyond this role in progression through the reaction cycle, it has been suggested that enzyme dynamics may also promote the chemical step itself. Dihydrofolate reductase (DHFR) is a model enzyme for which dynamics have been proposed to aid in both substrate flux and catalysis. The G121V mutant of DHFR is a well studied form that exhibits a severe reduction in the rate of hydride transfer yet there remains dispute as to whether this defect is caused by altered structure, dynamics, or both. Here we address this by presenting an NMR study of the G121V mutant bound to reduced cofactor and the transition state inhibitor, methotrexate. NMR chemical shift markers demonstrate that this form predominantly adopts the closed conformation thereby allowing us to provide the first glimpse into the dynamics of a catalytically relevant complex. Based on 15N and 2H NMR spin relaxation, we find that the mutant complex has modest changes in ps-ns flexibility with most affected residues residing in the distal adenosine binding domain rather than the active site. Thus, aberrant ps-ns dynamics are likely not the main contributor to the decreased catalytic rate. The most dramatic effect of the mutation involves changes in µs-ms dynamics of the F-G and Met20 loops. Whereas loop motion is quenched in the wild type transition state inhibitor complex, the F-G and Met20 loops undergo excursions from the closed conformation in the mutant complex. These excursions serve to decrease the population of conformers having the correct active site configuration, thus providing an explanation for the G121V catalytic defect

NMR Metabolomics Protocols for Drug Discovery

Drug discovery is an extremely difficult and challenging endeavor with a very high failure rate. The task of identifying a drug that is safe, selective and effective is a daunting proposition because disease biology is complex and highly variable across patients. Metabolomics enables the discovery of disease biomarkers, which provides insights into the molecular and metabolic basis of disease and may be used to assess treatment prognosis and outcome. In this regard, metabolomics has evolved to become an important component of the drug discovery process to resolve efficacy and toxicity issues, and as a tool for precision medicine. A detailed description of an experimental protocol is presented that outlines the application of NMR metabolomics to the drug discovery pipeline. This includes: (1) target identification by understanding the metabolic dysregulation in diseases, (2) predicting the mechanism of action of newly discovered or existing drug therapies, (3) and using metabolomics to screen a chemical lead to assess biological activity. Unlike other OMICS approaches, the metabolome is “fragile”, and may be negatively impacted by improper sample collection, storage and extraction procedures. Similarly, biologically-irrelevant conclusions may result from incorrect data collection, pre-processing or processing procedures, or the erroneous use of univariate and multivariate statistical methods. These critical concerns are also addressed in the protocol

Microstructure and mechanical properties of cryorolled aluminum alloy AA2219 in different thermomechanical processing conditions

In the present study, aluminum alloy AA2219-T87 bars were cryorolled to various amounts of deformation in two pre-deformation conditions: (1) without solution treatment i.e., as-received T87 (WST-CR) and (2) with solution treatment (ST + CR). The solution treated and cryorolled bars were further annealed leading to a third condition: (3) solution treated, cryorolled, and annealed (CR + Annealed). Room-temperature mechanical properties have been evaluated for all three cryorolled conditions. Significant improvement in the 0.2 pct YS and UTS values was obtained for bars cryorolled to cross-sectional area reduction of more than 50 pct in the solution-treated condition (ST + CR), whereas for bars cryorolled in the without solution-treated condition (WST-CR), only an improvement in the 0.2 pct YS was observed. Cryorolling did not enhance the precipitation kinetics nor did it increase the response of the alloy to aging. The mechanical properties were correlated to the microstructures obtained by optical and transmission electron microscopy. Microstructural evolution in the ST + CR condition indicated gradual progression of the principal restoration mechanism from dynamic recovery (DRV) to dynamic recrystallization with an increasing amount of plastic deformation. Transmission electron microscopy of WST-CR and ST + CR specimens showed an increase in dislocation density as a function of the amount of deformation indicating suppression of DRV at cryogenic temperatures. Cryorolling in the solution-treated condition to cross-sectional area reduction of more than 50 pct (ST + 70 pct CR) was found to impart an optimum combination of strength and percent elongation in the present study.by Aditya Sarkar, K. Saravanan, Niraj Nayan, S. V. S. Narayana Murty, P. Ramesh Narayanan, P. V. Venkitakrishnan and Jyoti Mukhopadhya

The Role of Large-Scale Motions in Catalysis by Dihydrofolate Reductase

Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR