Stereospecificity of hydride transfer and molecular docking in FMN-dependent NADH-indigo reductase of Bacillus smithii.
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ABSTRACT:
SUBMITTER: Yoneda K
PROVIDER: S-EPMC8255831 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:This paper reviews the results from hybrid quantum/classical molecular dynamics simulations of the hydride transfer reaction catalysed by wild-type (WT) and mutant Escherichia coli and WT Bacillus subtilis dihydrofolate reductase (DHFR). Nuclear quantum effects such as zero point energy and hydrogen tunnelling are significant in these reactions and substantially decrease the free energy barrier. The donor-acceptor distance decreases to ca 2.7 A at transition-state configurations to enable the hydride transfer. A network of coupled motions representing conformational changes along the collective reaction coordinate facilitates the hydride transfer reaction by decreasing the donor-acceptor distance and providing a favourable geometric and electrostatic environment. Recent single-molecule experiments confirm that at least some of these thermally averaged equilibrium conformational changes occur on the millisecond time-scale of the hydride transfer. Distal mutations can lead to non-local structural changes and significantly impact the probability of sampling configurations conducive to the hydride transfer, thereby altering the free-energy barrier and the rate of hydride transfer. E. coli and B. subtilis DHFR enzymes, which have similar tertiary structures and hydride transfer rates with 44% sequence identity, exhibit both similarities and differences in the equilibrium motions and conformational changes correlated to hydride transfer, suggesting a balance of conservation and flexibility across species.
| S-EPMC1647314 | biostudies-literature
Project description:DHFR (dihydrofolate reductase) catalyses the metabolically important reduction of 7,8-dihydrofolate by NADPH. DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR), which shares similarity with DHFR from Escherichia coli, has previously been characterized structurally. Its tertiary structure is similar to that of DHFR from E. coli but it is the only DHFR characterized so far that relies on dimerization for stability. The midpoint of the thermal unfolding of TmDHFR was at approx. 83 degrees C, which was 30 degrees C higher than the melting temperature of DHFR from E. coli. The turnover and the hydride-transfer rates in the kinetic scheme of TmDHFR were derived from measurements of the steady-state and pre-steady-state kinetics using absorbance and stopped-flow fluorescence spectroscopy. The rate constant for hydride transfer was found to depend strongly on the temperature and the pH of the solution. Hydride transfer was slow (0.14 s(-1) at 25 degrees C) and at least partially rate limiting at low temperatures but increased dramatically with temperature. At 80 degrees C the hydride-transfer rate of TmDHFR was 20 times lower than that observed for the E. coli enzyme at its physiological temperature. Hydride transfer depended on ionization of a single group in the active site with a p K(a) of 6.0. While at 30 degrees C, turnover of substrate by TmDHFR was almost two orders of magnitude slower than by DHFR from E. coli; the steady-state rates of the two enzymes differed only 8-fold at their respective working temperatures.
| S-EPMC1223599 | biostudies-other
Project description:NADH:ubiquinone oxidoreductase (complex I) is a complicated respiratory enzyme that conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. During catalysis, NADH oxidation by a flavin mononucleotide is followed by electron transfer to a chain of iron-sulfur clusters. Alternatively, the flavin may be reoxidized by hydrophilic electron acceptors, by artificial electron acceptors in kinetic studies, or by oxygen and redox-cycling molecules to produce reactive oxygen species. Here, we study two steps in the mechanism of NADH oxidation by complex I. First, molecular fragments of NAD(H), tested as flavin-site inhibitors or substrates, reveal that the adenosine moiety is crucial for binding. Nicotinamide-containing fragments that lack the adenosine do not bind, and ADP-ribose binds more strongly than NAD(+), suggesting that the nicotinamide is detrimental to binding. Second, the primary kinetic isotope effects from deuterated nicotinamide nucleotides confirm that hydride transfer is from the pro-S position and reveal that hydride transfer, along with NAD(+) dissociation, is partially rate-limiting. Thus, the transition state energies are balanced so that no single step in NADH oxidation is completely rate-limiting. Only at very low NADH concentrations does weak NADH binding limit NADH:ubiquinone oxidoreduction, and at the high nucleotide concentrations of the mitochondrial matrix, weak nucleotide binding constants assist product dissociation. Using fast nucleotide reactions and a balance between the nucleotide binding constants and concentrations, complex I combines fast and energy-conserving NADH oxidation with minimal superoxide production from the nucleotide-free site.
| S-EPMC3680915 | biostudies-literature
Project description:Hydride transfer is widespread in nature and has an essential role in applied research. However, the mechanisms of how this transformation occurs in living organisms remain a matter of vigorous debate. Here, we examined dihydrofolate reductase (DHFR), an enzyme that catalyzes hydride from C4' of NADPH to C6 of 7,8-dihydrofolate (H2F). Despite many investigations of the mechanism of this reaction, the contribution of polarization of the ?-bond of H2F in driving hydride transfer remains unclear. H2F was stereospecifically labeled with deuterium ? to the reacting center, and ?-deuterium kinetic isotope effects were measured. Our experimental results combined with analysis derived from QM/MM simulations reveal that hydride transfer is triggered by polarization at the C6 of H2F. The ? C?-H bonds contribute to the buildup of the cationic character during the chemical transformation, and hyperconjugation influences the formation of the transition state. Our findings provide key insights into the hydride transfer mechanism of the DHFR-catalyzed reaction, which is a target for antiproliferative drugs and a paradigmatic model in mechanistic enzymology.
| S-EPMC7007191 | biostudies-literature
Project description:Morphinone reductase (MR) is an important model system for studying the contribution of protein motions to H-transfer reactions. In this research, we used quantum mechanical/molecular mechanics (QM/MM) simulation together with transition path sampling (TPS) simulation to study two important topics of current research on MR: the existence of multiple catalytic reaction pathways and the involvement of fast protein motions in the catalytic process. We have discovered two reaction pathways for the wild type and three reaction pathways for the N189A mutant. With the committor distribution analysis method, we found reaction coordinates for all five reaction pathways. Only one wild-type reaction pathway has a rate-promoting vibration from His186, while all of the other four pathways do not involve any protein motions in their catalytic process through the transition state. The rate-promoting vibration in the wild-type MR, which comes from a direction perpendicular to the donor-acceptor axis, functions to decrease the donor-acceptor distance by causing a subtle "out-of-plane" motion of a donor atom. By comparing reaction pathways between the two enzymes, we concluded that the major effect of the N189A point mutation is to increase the active site volume by altering the active site backbone and eliminating the Asn189 side chain. This effect causes a different NADH geometry at the reactant state, which very well explains the different reaction mechanisms between the two enzymes, as well as the disappearance of the His186 rate-promoting vibrations in the N189A mutant. The unfavorable geometry of the NADH pyridine ring induced by the N189A point mutation is the potential cause of multiple reaction pathways in N189A mutants.
| S-EPMC7496013 | biostudies-literature
Project description:Complex I functions as an initial electron acceptor in aerobic respiratory chains that reduces quinone and pumps protons across a biological membrane. This remarkable charge transfer process extends ca. 300 Å and it is initiated by a poorly understood proton-coupled electron transfer (PCET) reaction between nicotinamide adenine dinucleotide (NADH) and a protein-bound flavin (FMN) cofactor. We combine here large-scale density functional theory calculations and quantum/classical models with atomistic molecular dynamics simulations to probe the energetics and dynamics of the NADH-driven PCET reaction in complex I. We find that the reaction takes place by concerted hydrogen atom (H•) transfer that couples to an electron transfer (eT) between the aromatic ring systems of the cofactors and further triggers reduction of the nearby FeS centers. In bacterial, Escherichia coli-like complex I isoforms, reduction of the N1a FeS center increases the binding affinity of the oxidized NAD+ that prevents the nucleotide from leaving prematurely. This electrostatic trapping could provide a protective gating mechanism against reactive oxygen species formation. We also find that proton transfer from the transient FMNH• to a nearby conserved glutamate (Glu97) residue favors eT from N1a onward along the FeS chain and modulates the binding of a new NADH molecule. The PCET in complex I isoforms with low-potential N1a centers is also discussed. On the basis of our combined results, we propose a putative mechanistic model for the NADH-driven proton/electron-transfer reaction in complex I.
| S-EPMC6890364 | biostudies-literature
Project description:Ferredoxin: NADP+ reductase (FNR) is an FAD-containing enzyme best known for catalysing the transfer of electrons from ferredoxin (Fd) to NADP+ to make NADPH during photosynthesis. It is also the prototype for a broad enzyme superfamily, including the NADPH oxidases (NOXs) that all catalyse similar FAD-enabled electron transfers between NAD(P)H and one-electron carriers. Here, we define further mechanistic details of the NAD(P)H ? FAD hydride-transfer step of the reaction based on spectroscopic studies and high-resolution (~ 1.5 Å) crystallographic views of the nicotinamide-flavin interaction in crystals of corn root FNR Tyr316Ser and Tyr316Ala variants soaked with either nicotinamide, NADP+ , or NADPH. The spectra obtained from FNR crystal complexes match those seen in solution and the complexes reveal active site packing interactions and patterns of covalent distortion of the FAD that imply significant active site compression that would favour catalysis. Furthermore, anisotropic B-factors show that the mobility of the C4 atom of the nicotinamide in the FNR:NADP+ complex has a directionality matching that expected for boat-like excursions of the nicotinamide ring thought to enhance hydride transfer. Arguments are made for the relevance of this binding mode to catalysis, and specific consideration is given to how the results extrapolate to provide insight to structure-function relations for the membrane-bound NOX enzymes for which little structural information has been available. DATABASES:Structural data are available in the PDB database under the accession numbers 3LO8 (wild-type), 5VW4 [Y316S:nicotinamide (P32 21)], 5VW9 [Y316S:nicotinamide (P31 21)], 5VW3 [Y316S:NADP+ (P32 21)], 5VW8 [Y316S:NADP+ (P31 21)], 5VW2 [Y316S:NADPH (P32 21)], 5VW5 [Y316A:nicotinamide (P32 21)], 5VW6 [Y316A:NADP+ (P32 21)], 5VW7 [Y316A:NADPH (P32 21)], 5VWA [Y316F (P32 21)], and 5VWB [Y316F:NADP+ (P31 21)]. Enzyme Commission number: ferredoxin:NADP+ reductase - E C1.18.1.2.
| S-EPMC5626627 | biostudies-literature
Project description:Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the "140s" FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the "140s loop" by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the "140s loop".
| S-EPMC8508823 | biostudies-literature
Project description:The catalytic cycle of an enzyme is frequently associated with conformational changes that may limit maximum catalytic throughput. In Escherichia coli dihydrofolate reductase, release of the tetrahydrofolate (THF) product is the rate-determining step under physiological conditions and is associated with an "occluded" to "closed" conformational change. In this study, we demonstrate that in dihydrofolate reductase the closed to occluded conformational change in the product ternary complex (E.THF.NADP (+)) also gates progression through the catalytic cycle. Using NMR relaxation dispersion, we have measured the temperature and pH dependence of microsecond to millisecond time scale backbone dynamics of the occluded E.THF.NADP (+) complex. Our studies indicate the presence of three independent dynamic regions, associated with the active-site loops, the cofactor binding cleft, and the C-terminus and an adjacent loop, which fluctuate into discrete conformational substates with different kinetic and thermodynamic parameters. The dynamics of the C-terminally associated region is pH-dependent (p K a < 6), but the dynamics of the active-site loops and cofactor binding cleft are pH-independent. The active-site loop dynamics access a closed conformation, and the accompanying closed to occluded rate constant is comparable to the maximum pH-independent hydride transfer rate constant. Together, these results strongly suggest that the closed to occluded conformational transition in the product ternary complex is a prerequisite for progression through the catalytic cycle and that the rate of this process places an effective limit on the maximum rate of the hydride transfer step.
| S-EPMC2562322 | biostudies-literature
Project description:HMG-CoA reductase catalyzes the four-electron reduction of HMG-CoA to mevalonate and is an enzyme of considerable biomedical relevance because of the impact of its statin inhibitors on public health. Although the reaction has been studied extensively using X-ray crystallography, there are surprisingly no computational studies that test the mechanistic hypotheses suggested for this complex reaction. Theozyme and quantum mechanical (QM)/molecular mechanical (MM) calculations up to the B3LYP/6-31g(d,p)//B3LYP/6-311++g(2d,2p) level of theory were employed to generate an atomistic description of the enzymatic reaction process and its energy profile. The models generated here predict that the catalytically important Glu83 is protonated prior to hydride transfer and that it acts as the general acid or base in the reaction. With Glu83 protonated, the activation energies calculated for the sequential hydride transfer reactions, 21.8 and 19.3 kcal/mol, are in qualitative agreement with the experimentally determined rate constant for the entire reaction (1 s(-1) to 1 min(-1)). When Glu83 is not protonated, the first hydride transfer reaction is predicted to be disfavored by >20 kcal/mol, and the activation energy is predicted to be higher by >10 kcal/mol. While not involved in the reaction as an acid or base, Lys267 is critical for stabilization of the transition state in forming an oxyanion hole with the protonated Glu83. Molecular dynamics simulations and MM/Poisson-Boltzmann surface area free energy calculations predict that the enzyme active site stabilizes the hemithioacetal intermediate better than the aldehyde intermediate. This suggests a mechanism in which cofactor exchange occurs before the breakdown of the hemithioacetal. Slowing the conversion to aldehyde would provide the enzyme with a mechanism to protect it from solvent and explain why the free aldehyde is not observed experimentally. Our results support the hypothesis that the pK(a) of an active site acidic group is modulated by the redox state of the cofactor. The oxidized cofactor and deprotonated Glu83 are closer in space after hydride transfer, indicating that indeed the cofactor may influence the pK(a) of Glu83 through an electrostatic interaction. The enzyme is able to catalyze the transfer of a hydride to the structurally and electronically distinct substrates by maintaining the general shape of the active site and adjusting the electrostatic environment through acid-base chemistry. Our results are in good agreement with the well-studied hydride transfer reactions catalyzed by liver alcohol dehydrogenase in calculated energy profile and reaction geometries despite different mechanistic functionalities.
| S-EPMC3522576 | biostudies-literature