One site fits both: A model for the ternary complex of folate + NADPH in R67 dihydrofolate reductase, a D2 symmetric enzyme

R67 dihydrofolate reductase (DHFR) is a novel enzyme that confers resistance to the antibiotic trimethoprim. The crystal structure of R67 DHFR displays a toroidal structure with a central active-site pore. This homotetrameric protein exhibits 222 symmetry, with only a few residues from each chain contributing to the active site, so related sites must be used to bind both substrate (dihydrofolate) and cofactor (NADPH) in the productive R67 DHFR?NADPH?dihydrofolate complex. Whereas the site of folate binding has been partially resolved crystallographically, an interesting question remains: how can the highly symmetrical active site also bind and orient NADPH for catalysis? To model this ternary complex, we employed DOCK and SLIDE, two methods for docking flexible ligands into proteins using quite different algorithms. The bound pteridine ring of folate (Fol I) from the crystal structure of R67 DHFR was used as the basis for docking the nicotinamide-ribose-P_i (NMN) moiety of NADPH. NMN was positioned by both DOCK and SLIDE on the opposite side of the pore from Fol I, where it interacts with Fol I at the pore's center. Numerous residues serve dual roles in binding. For example, Gln 67 from both the B and D subunits has several contacts with the pteridine ring, while the same residue from the A and C subunits has several contacts with the nicotinamide ring. The residues involved in dual roles are generally amphipathic, allowing them to make both hydrophobic and hydrophilic contacts with the ligands. The result is a `hot spot' binding surface allowing the same residues to co-optimize the binding of two ligands, and orient them for catalysis.     

Preorganization and protein dynamics in enzyme catalysis

Recently, an alternative has been offered to the concept of transition state (TS) stabilization as an explanation for rate enhancements in enzyme-catalyzed reactions. Instead, most of the rate increase has been ascribed to preorganization of the enzyme active site to bind substrates in a geometry close to that of the TS, which then transit the activation barrier impelled by motions along the reaction coordinate. The question as to how an enzyme achieves such preorganization and concomitant TS stabilization as well as potential coupled motions along the reaction coordinate leads directly to the role of protein dynamic motion. Dihydrofolate reductase (DHFR) is a paradigm in which the role of dynamics in catalysis continues to be unraveled by a wealth of kinetic, structural, and computational studies. DHFR has flexible loop regions adjacent to the active site whose motions modulate passage through the kinetically preferred pathway. The participation of residues distant from the DHFR active site in enhancing the rate of hydride transfer, however, is unanticipated and may signify the importance of long range protein motions. The general significance of protein dynamics in understanding other biological processes is briefly discussed.     

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.     

Isotachophoretic analysis of the dihydrofolate reductase reaction in the presence of methotrexate and ascorbic acid

The antifolate methotrexate (MTX) is widely used in cancer chemotherapy. In this study, we show that MTX (MTX-Glu1) and MTX-polyglutamates (MTX-Glu2-5) strongly inhibited the growth of the leukemic cell line MOLT-4. This effect, however, was mitigated by ascorbic acid. We investigated whether ascorbic acid is able to reduce dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) directly or by circumventing the MTX inhibition of dihydrofolate reductase (DHFR). The inhibition of this NADPH-dependent reduction of DHF by MTX-Glun in the absence or presence of ascorbate, was determined by analytical isotachophoresis. Using 0.01 M HCl/histidine, pH 6.0, as a leading electrolyte (L) and 0.005 M 2-(N-morpholino)ethanesulfonic acid (MES)/histidine, pH 6.0, as a terminating electrolyte (T), MTX-Glun derivatives including MTX-Glu1 could be easily separated, whereas the quantitative estimation of THF was not possible. A quantitative characterization of the DHFR reaction by measuring NADPH, NADP+ and ascorbate was achieved with another system (L: 0.01 M HCI/beta-alanine, pH 3.73; T: 0.01 M caproic acid, pH 3.27). Nanomolar concentrations of MTX-Glu1-5 inhibited consumption of NADPH and production of NADP+. Ascorbic acid was not able to reduce DHF, neither directly nor after inhibition of DHFR by MTX. However, ascorbic acid seemed to diminish the oxidation of THF and this may account for its capacity to reduce the inhibitory effect of MTX on MOLT-4 cells.     

Self-assembled enzymatic monolayer directly bound to a gold surface: activity and molecular recognition force spectroscopy studies

Escherichia coli dihydrofolate reductase (ecDHFR) has one surface cysteine, C152, located opposite and distal to the active site. Here, we show that the enzyme spontaneously assembles on an ultraflat gold surface as a homogeneous, covalently bound monolayer. Surprisingly, the activity of the gold-immobilized ecDHFR as measured by radiographic analysis was found to be similar to that of the free enzyme in solution. Molecular recognition force spectroscopy was used to study the dissociation forces involved in the rupture of AFM probe-tethered methotrexate (MTX, a tight-binding inhibitor of DHFR) from the gold-immobilized enzyme. Treatment of the ecDHFR monolayer with free MTX diminished the interaction of the functionalized tip with the surface, suggesting that the interaction was indeed active-site specific. These findings demonstrate the viability of a simple and direct enzymatic surface-functionalization without the use of spacers, thus, opening the door to further applications in the area of biomacromolecular force spectroscopy.     

A molecular model of the folate binding site of Pneumocystis carinii dihydrofolate reductase

Summary The inhibition of Pneumocystis carinii dihydrofolate reductase (DHFR) continues to be the major treatment strategy for P. carinii pneumonia (PCP). The design of new anti-pneumocystis agents would be significantly enhanced by the availability of a 3D model of the methotrexate (MTX) binding site of the P. carinii DHFR. However, an X-ray crystal structure of the P. carinii DHFR is not yet available. Alignment of the amino acid sequences of P. carinii and Lactobacillus casei DHFRs indicates that the two proteins show approximately 80% homology among MTX binding-site residues. This high level of homology suggests that the L. casei DHFR MTX binding-site structure could serve as a structural template in developing a model of the P. carinii DHFR MTX binding site. Therefore, the X-ray crystal structure of L. casei DHFR was used to develop a 3D model of the methotrexate binding site of P. carinii DHFR. The molecular modeling and dynamics software QUANTA/CHARMm was used. Amino acid residue mutations and deletions were performed using QUANTA and macromolecular minimizations were achieved with CHARMm. The MTX binding-site residues of L. casei DHFR were mutated to the corresponding residues of the P. carinii DHFR sequence. The resulting structure was extensively minimized. The resulting P. carinii MTX binding-site model showed significant differences in hydrogen-bonding patterns from the L. casei MTX binding site. Also, the P. carinii site is more hydrophobic than the corresponding L. casei site. Analysis of atom-to-atom close contacts between methotrexate and protein binding-site residues indicates that the P. carinii MTX binding-site complex is primarily stabilized by hydrophobic interactions, while the L. casei complex is mostly stabilized by electrostatic interactions. The model is consistent with the observed increased sensitivity of P. carinii DHFR to lipid-soluble inhibitors and provides a rational basis for the design of new anti-pneumocystis agents.     

Excited‐State Charge Separation in the Photochemical Mechanism of the Light‐Driven Enzyme Protochlorophyllide Oxidoreductase

The unique light‐driven enzyme protochlorophyllide oxidoreductase (POR) is an important model system for understanding how light energy can be harnessed to power enzyme reactions. The ultrafast photochemical processes, essential for capturing the excitation energy to drive the subsequent hydride‐ and proton‐transfer chemistry, have so far proven difficult to detect. We have used a combination of time‐resolved visible and IR spectroscopy, providing complete temporal resolution over the picosecond–microsecond time range, to propose a new mechanism for the photochemistry. Excited‐state interactions between active site residues and a carboxyl group on the Pchlide molecule result in a polarized and highly reactive double bond. This so‐called “reactive” intramolecular charge‐transfer state creates an electron‐deficient site across the double bond to trigger the subsequent nucleophilic attack of NADPH, by the negatively charged hydride from nicotinamide adenine dinucleotide phosphate. This work provides the crucial, missing link between excited‐state processes and chemistry in POR. Moreover, it provides important insight into how light energy can be harnessed to drive enzyme catalysis with implications for the design of light‐activated chemical and biological catalysts.     

A theoretical study of the catalytic mechanism of formate dehydrogenase

A theoretical study of the hydride transfer between formate anion and nicotinamide adenine dinucleotide (NAD(+)) catalyzed by the enzyme formate dehydrogenase (FDH) has been carried out by a combination of two hybrid quantum mechanics/molecular mechanics techniques: statistical simulation methods and internal energy minimizations. Free energy profiles, obtained for the reaction in the enzyme active site and in solution, allow obtaining a comparative analysis of the behavior of both condensed media. Moreover, calculations of the reaction in aqueous media can be used to probe the dramatic differences between reactants state in the enzyme active site and in solution. The results suggest that the enzyme compresses the substrate and the cofactor into a conformation close to the transition structure by means of favorable interactions with the amino acid residues of the active site, thus facilitating the relative orientation of donor and acceptor atoms to favor the hydride transfer. Moreover, a permanent field created by the protein reduces the work required to reach the transition state (TS) with a concomitant polarization of the cofactor that would favor the hydride transfer. In contrast, in water the TS is destabilized with respect to the reactant species because the polarity of the solute diminishes as the reaction proceeds, and consequently the reaction field, which is created as a response to the change in the solute polarity, is also decreased. Therefore protein structure is responsible of both effects; substrate preorganization and TS stabilization thus diminishing the activation barrier. Because of the electrostatic features of the catalyzed reaction, both media preferentially stabilize the ground-state, thus explaining the small rate constant enhancement of this enzyme, but FDH does so to a much lower extent than aqueous solution. Finally, a good agreement between experimental and theoretical kinetic isotope effects is found, thus giving some credit to our results.     

Multiple ligand-binding modes in bacterial R67 dihydrofolate reductase

Summary R67 dihydrofolate reductase (DHFR), a bacterial plasmid-encoded enzyme associated with resistance to the drug trimethoprim, shows neither sequence nor structural homology with the chromosomal DHFR. It presents a highly symmetrical toroidal structure, where four identical monomers contribute to the unique central active-site pore. Two reactants (dihydrofolate, DHF), two cofactors (NADPH) or one of each (R67•DHF•NADPH) can be found simultaneously within the active site, the last one being the reactive ternary complex. As the positioning of the ligands has proven elusive to empirical determination, we addressed the problem from a theoretical perspective. Several potential structures of the ternary complex were generated using the docking programs AutoDock and FlexX. The variability among the final poses, many of which conformed to experimental data, prompted us to perform a comparative scoring analysis and molecular dynamics simulations to assess the stability of the complexes. Analysis of ligand–ligand and ligand–protein interactions along the 4 ns trajectories of eight different structures allowed us to identify important inter-ligand contacts and key protein residues. Our results, combined with published empirical data, clearly suggest that multipe binding modes of the ligands are possible within R67 DHFR. While the pterin ring of DHF and the nicotinamide ring of NADPH assume a stacked endo-conformation at the centre of the pore, probably assisted by V66, Q67 and I68, the tails of the molecules extend towards opposite ends of the cavity, adopting multiple configurations in a solvent rich-environment where hydrogen-bond interactions with K32 and Y69 may play important roles.     

Mechanistic aspects of biological redox reactions involving NADH 2: A combined semiempirical and ab initio study of hydride-ion transfer between the NADH analogue, 1-methyl-dihydronicotinamide,and folate and dihydrofolate analogue substrates of dihydrofolate reductase

As part of a study of factors controlling biological redox reactions of nicotinamide cofactors [nicotinamide adenine dinucleotide (phosphate) NAD(P)H], we have investigated the effect on a model reaction of the conformational state (cis or trans) of the carboxamide side chain, using quantum chemical methods. The reaction is that for the enzyme dihydrofolate reductase between the NADPH analogue, 1-methyl-dihydronicotinamide, and the protonated forms of the folate and dihydrofolate substrate analogues, pyrazine and dihydropyrazine. Some calculations on pterin and dihydropterin substrate analogues were also carried out in order to gauge the effects of inter-ring coupling. The influence of carboxamide side-chain conformation of nicotinamide on the energetics of the hydride-ion transfer, and on the structures of the transition states and stable intermolecular-interaction complexes, are examined as a function of the orientation of approach of the reactants. These approach geometries include those corresponding to the observed binding of cofactor and either substrate or inhibitor in the enzyme active site. Reactant, product, reactants-complex, and transition-state geometries were optimized at the semiempirical AM1 level, while ab initio SCF/STO-3G and SCF/3-21G single-point calculations were carried out at the AM1 optimized geometries for all species, as well as full geometry optimizations for isolated reactants and products. The results show that reactants-complex and transition-state energies are lower for the trans conformer of dihydronicotinamide than for the cis conformer, due to more favorable H-bonding or electrostatic interactions with the protonated substrate. Also, consideration of the structural parameters, including reaction coordinate bond lengths, ring geometries, and charge distributions, indicate that the trans transition states are more product-like than those for the cis. For the (trans) approaches corresponding to the enzymic orientation for substrate, the intermolecular interaction for the folate reaction lacks the stabilizing influence of the formal H-bond which is present for the dihydrofolate reaction, and consequently the reactants-complex and transition state are less stable.     

Comparison of coupled motions in Escherichia coli and Bacillus subtilis dihydrofolate reductase

Hybrid quantum/classical molecular dynamics simulations are used to compare the role of protein motion in the hydride transfer reaction catalyzed by Escherichia coli and Bacillus subtilis dihydrofolate reductase (DHFR). These two enzymes have 44% sequence identity, and the experimentally determined structures and hydride transfer rates are similar. The simulations indicate that the tertiary structures of both enzymes evolve in a similar manner during the hydride transfer reaction. In both enzymes, the donor-acceptor distance decreases to approximately 2.7 Angstroms at the transition state configurations to enable hydride transfer. Zero point energy and hydrogen tunneling effects are found to be significant for both enzymes. Covariance and rank correlation analyses of motions throughout the protein and ligands illustrate that E. coli and B. subtilis DHFR exhibit both similarities and differences in the equilibrium fluctuations and the conformational changes along the collective reaction coordinate for hydride transfer. A common set of residues that play a significant role in the network of coupled motions leading to configurations conducive to hydride transfer for both E. coli and B. subtilis DHFR was identified. These results suggest a balance between conservation and flexibility in the thermal motions and conformational changes during hydride transfer. Homologous protein structures, in conjunction with conformational sampling, enable enzymes with different sequences to catalyze the same hydride transfer reaction with similar efficiency.     

A proposed model of Mycobacterium avium complex dihydrofolate reductase and its utility for drug design

A homology model of Mycobacterium avium complex dihydrofolate reductase (MAC DHFR) was constructed on the basis of the X-ray crystal structure of Mycobacterium tuberculosis (Mtb) DHFR. The homology searching of the MAC DHFR resulted in the identification of the Mtb DHFR structure (PDB 1DF7) as the template for the model building. The MAC enzyme sequence was aligned to that of the Mtb counterpart using a modified Needleman and Wunsch methodology. The initial geometry to be modeled was copied from the template, either fully or partially depending on whether the residues were conserved or not, respectively. Using a randomized modeling procedure, 10 independent models of the target protein were built. The cartesian average of all the model structures was then refined using molecular mechanics. The resulting model was assessed for stereochemical quality using a Ramachandran plot and by analyzing the consistency of the model with the experimental data. The structurally and functionally important residues were identified from the model. Further, 5-deazapteridines recently reported as inhibitors of MAC DHFR were docked into the active site of the developed model. All the seven inhibitors used in the docking study have a similar docking mode at the active site. The network of hydrogen bonds around the 2,4-diamino-5-deazapteridine ring was found to be crucial for the binding of the inhibitors with the active site residues. The 5-methyl group of the inhibitors was located in a narrow hydrophobic pocket at the bottom of the active site. The relative values of the three torsion angles of the inhibitors were found to be important for the proper orientation of the inhibitor functional groups into the active site.     

Simulations of remote mutants of dihydrofolate reductase reveal the nature of a network of residues coupled to hydride transfer

Recent experimental and theoretical studies have proposed that enzymes involve networks of coupled residues throughout the protein that participate in motions accompanying chemical barrier crossing. Here, we have examined portions of a proposed network in dihydrofolate reductase (DHFR) using quantum mechanics/molecular mechanics simulations. The simulations use a hybrid quantum mechanics‐molecular mechanics approach with a recently developed semiempirical AM1‐SRP Hamiltonian that provides accurate results for this reaction. The simulations reproduce experimentally determined catalytic rates for the wild type and distant mutants of E. coli DHFR, underscoring the accuracy of the simulation protocol. Additionally, the simulations provide detailed insight into how residues remote from the active site affect the catalyzed chemistry, through changes in the thermally averaged properties along the reaction coordinate. The mutations do not greatly affect the structure of the transition state near the bond activation, but we observe differences somewhat removed from the point of C? H cleavage that affect the rate. The mutations have global effects on the thermally averaged structure that propagate throughout the enzyme and the current simulations highlight several interactions that appear to be particularly important. © 2014 Wiley Periodicals, Inc.     

Conformational substates modulate hydride transfer in dihydrofolate reductase

In earlier studies of the hydride-transfer reaction catalyzed by dihydrofolate reductase (DHFR) we identified features of the protein correlated with variations in the reaction barrier. We extend the scope of those studies by carrying out potential of mean force (PMF) simulations to determine the hydride-transfer barrier in the wild-type protein as well as the G121V and G121S mutants. While our prior studies focused on the reactant state, our current work addresses the full reaction pathway and directly probes the reactive event. The free energy barriers and structural ensembles resulting from these PMF calculations exhibit the same trends reported in our previous work. Fluctuations present in these simulations also exhibit trends associated with differences in the hydride-transfer barrier height. Moreover, vibrational modes anticipated to promote hydride transfer exhibit larger amplitudes in simulations that generate lowered barriers. The results of our study indicate that discrete basins (substates) on a potential energy landscape of the enzyme give rise to distinct hydride-transfer barriers. We suggest that the long-range effects of mutations at position 121 within DHFR are mediated by differentially preorganized protein environments in the context of distinct substate distributions, with concomitant changes to the dynamic properties of the enzyme.     

How dihydrofolate reductase facilitates protonation of dihydrofolate

Dihydrofolate Reductase (DHFR) catalyzes the reduction of dihydrofolate (H2F) to tetrahydrofolate. On the basis of 10-12.5 ns molecular dynamics simulations of two conformations (closed and occluded) of the ternary DHFR/NADPH/H2F complex from Escherichia coli and a free energy perturbation approach, we have calculated the pKa value for the N5 atom in H2F. Our results suggest that the N5 atom in H2F is responsible for the pH dependency of the catalyzed reaction, meaning that DHFR facilitates protonation of H2F by approximately 4 pKa units. The mechanism behind this increase is due to favorable electrostatic interactions between the Asp27 residue and a proton at the N5 atom. The electrostatic interactions are enhanced by a hydrophobic active site, which to a large extent is made hydrophobic by the M20 loop in DHFR. Moreover, we find that the conformation imposed on H2F by DHFR to some extent also favors protonation of the N5 atom. Our results add support to previous findings and suggestions by Callender and co-workers [e.g., Deng, J.; Callender, R. J. Am. Chem. Soc. 1998, 120, 7730-7737] and explain why mutation of Asp27 may lead to severely reduced activity at neutral pH.     

荧光光度法二氢叶酸还原酶抑制剂筛选模型的建立和应用

本文应用二氢叶酸(DHFA)和辅酶Ⅱ(NADPH)的荧光性质,建立了二氢叶酸还原酶(DHFR)活力测定的反应体系,并对酸度等反应条件进行了优化。在该体系中,用双倒数作图法分别测定了二氢叶酸还原酶对二氢叶酸和辅酶Ⅱ的表观米氏常数。将所建立的体系应用于氨甲喋呤,甲氧苄氨嘧啶等已知抑制剂的抑制率的测定,结果满意。     

Computational Study of Mechanism and Enantioselectivity of Imine Reductase from Amycolatopsis orientalis

Imine reductases (IREDs) are NADPH-dependent enzymes (NADPH=nicotinamide adenine dinucleotide phosphate) that catalyze the reduction of imines to amines. They exhibit high enantioselectivity for a broad range of substrates, making them of interest for biocatalytic applications. In this work, we have employed density functional theory (DFT) calculations to elucidate the reaction mechanism and the origins of enantioselectivity of IRED from Amycolatopsis orientalis. Two substrates are considered, namely 1-methyl-3,4-dihydroisoquinoline and 2-propyl-piperideine. A model of the active site is built on the basis of the available crystal structure. For both substrates, different binding modes are first evaluated, followed by calculation of the hydride transfer transition states from each complex. We have also investigated the effect of mutations of certain important active site residues (Tyr179Ala and Asn241Ala) on the enantioselectivity. The calculated energies are consistent with the experimental observations and the analysis of transition states geometries provides insights into the origins of enantioselectivity of this enzyme.     

二氢叶酸还原酶抑制剂的高效毛细管电泳法筛选模型的建立与应用

建立了一种采用毛细管电泳法(CE)测定二氢叶酸还原酶(DHFR)反应动力学参数的新方法。以含0.002%Brij-35的50 mmol/L 的硼砂缓冲溶液（pH 9.18）作为电泳介质,检测波长280 nm,于19 min内实现了体系中各组分的基线分离。根据酶反应过程中反应物和反应产物的浓度变化计算有关反应动力学参数。将已知的氨甲喋呤、甲氧苄胺嘧啶和叶酸3种抑制剂作用于所建立的二氢叶酸还原酶体系,测得抑制剂的半数抑制浓度与文献值相接近,证明本体系可用于二氢叶酸还原酶抑制剂(DHFRI)的筛选。     

An old but simple and efficient method to elucidate the oxidation mechanism of NAD(P)H model 1-Aryl-1,4-dihydronicotinamides by cations 2-methyl-5-nitroisoquinolium, tropylium, and xanthylium in aqueous solution.

Cations 2-methyl-5-nitroisoquinolinium (IQ+), tropylium (T+), and xanthylium (Xn+) were treated by an NAD(P)H model 1-(p-substituted phenyl)-1.4-dihydronicotinamide series (1) in buffered aqueous solution to give the corresponding reduced products by accepting hydride. Effects of the 4-substituents of 1 on the reaction rates were investigated. Hammett's linear free energy relationship analysis on the three reactions of 1 provides the reaction constants of -0.48, -2.2, and -1.4 with IQ+, T+, and Xn+ as the hydride acceptors, respectively. Comparison of the present reactions with the reaction examples whose mechanisms are well-known, such as the reaction of 1 with a one-electron oxidant Fe(CN)6(-3), shows that the active site of 1 in the oxidation with IQ+ is at the 4-position on the dihydropyridine ring but that the active site of 1 in the oxidations with T+ and Xn+ is at the 1-position, which is in agreement with the results from the Br?nsted-type linear analysis and the relation studies of the logarithm of the second-order rate constants with the oxidation potentials of the hydride donors. According to the dependence of the reaction mechanism on the active site of 1, a conclusion can be made that the reaction of 1 with IQ+ proceeds by direct one-step hydride transfer mechanism, but the reactions of 1 with T+ and Xn+ would take place via multistep hydride transfer mechanism initiated by one-electron transfer.     

Computational algorithmic and molecular dynamics study of functional and structural impacts of non-synonymous single nucleotide polymorphisms in human DHFR gene

Human dihydrofolate reductase (DHFR) is a conserved enzyme that is central to folate metabolism and is widely targeted in pathogenic diseases as well as cancers. Although studies have reported the fact that genetic mutations in DHFR leads to a rare autosomal recessive inborn error of folate metabolism and drug resistance, there is a lack of an extensive study on how the deleterious non-synonymous SNPs (nsSNPs) disrupt its phenotypic effects. In this study, we aim at discovering the structural and functional consequences of nsSNPs in DHFR by employing a combined computational approach consisting of ten recently developed in silico tools for identification of damaging nsSNPs and molecular dynamics (MD) simulation for getting deeper insights into the magnitudes of damaging effects. Our study revealed the presence of 12 most deleterious nsSNPs affecting the native phenotypic effects, with three (R71T, G118D, Y122D) identified in the co-factor and ligand binding active sites. MD simulations also suggested that these three SNPs particularly Y122D, alter the overall structural flexibility and dynamics of the native DHFR protein which can provide more understandings into the crucial roles of these mutants in influencing the loss of DHFR function.