ABSTRACT
Gene silencing in bacteria is mediated by chromatin proteins, of which Escherichia coli H-NS is a paradigmatic example. H-NS forms nucleoprotein filaments with either one or two DNA duplexes. However, the structures, arrangements of DNA-binding domains (DBDs), and positions of DBD-DNA contacts in linear and bridged filaments are uncertain. To characterize the H-NS DBD contacts that silence transcription by RNA polymerase, we combined ·OH footprinting, molecular dynamics, statistical modeling, and DBD mapping using a chemical nuclease (Fe2+-EDTA) tethered to the DBDs (TEN-map). We find that H-NS DBDs contact DNA at indistinguishable locations in bridged or linear filaments and that the DBDs vary in orientation and position with â¼10-bp average spacing. Our results support a hemi-sequestration model of linear-to-bridged H-NS switching. Linear filaments able to inhibit only transcription initiation switch to bridged filaments able to inhibit both initiation and elongation using the same irregularly spaced DNA contacts.
ABSTRACT
The microscopic properties that determine hygroscopic behavior are complex. The importance of hygroscopicity to many areas, and particularly atmospheric chemistry, in terms of aerosol growth and cloud nucleation, mandate the need for robust models to understand this behavior. Toward this end, we have employed molecular dynamics simulations to calculate hygroscopicity from atomistic models using free energy perturbation. We find that currently available force fields may not be well-suited to modeling the extreme environments of aerosol particles. Nonetheless, the results illuminate some shortcomings in our current understanding of hygroscopic growth and cloud nucleation. The most widely used model of hygroscopicity, κ-Köhler Theory (κKT), breaks down in the case of deviations from ideal solution behavior and empirical adjustments within the simplified framework cannot account for non-ideal behavior. A revised model that incorporates non-ideal mixing rescues the general framework of κKT and allows us to understand our simulation results as well as the behavior of atmospheric aerosols over the full range of humidity. The revised model shows that non-ideal mixing dominates hygroscopic growth at subsaturation humidity. Thus, a model based on ideal mixing will fail to predict subsaturation growth from cloud condensation nucleus (CCN) activation or vice versa; a single parameter model for hygroscopicity will generally be insufficient to extrapolate across wide ranges of humidity. We argue that in many cases, when data are limited to subsaturation humidity, an empirical model for non-ideal mixing may be more successful than one for ideal mixing.
ABSTRACT
Transphosphorylation of nucleotide triphosphates is the central reaction in DNA replication by DNA polymerase as well as many other biological processes. Despite its importance, the microscopic chemical mechanism of transphosphorylation of nucleotide triphosphates is, in most cases, unknown. Here we use extensive simulations of DNA polymerase η to test mechanistic hypotheses. We systematically survey the reactive space by calculating 2D free-energy surfaces for 10 different plausible mechanisms that have been proposed. We supplement these free-energy surfaces with calculations of pKa for a number of potentially acidic protons in different states relevant to the catalytic cycle. We find that among all of the conditions that we test, the smallest activation barrier occurs for a reaction where a Mg2+-coordinated water deprotonates the nucleophilic 3'-OH, and this deprotonation is concerted with the phosphoryl transfer. The presence of a third Mg2+ in the active site lowers the activation barrier for the water-as-base mechanism, as does protonation of the pyrophosphate leaving group, which is consistent with general acid catalysis. The results demonstrate the value of simulations, when used in conjunction with experimental data, to help establish a microscopic chemical mechanism in a complex environment.
Subject(s)
DNA-Directed DNA Polymerase , Nucleotides , Water , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , Magnesium/chemistry , Magnesium/metabolism , Metabolic Networks and Pathways , Molecular Dynamics Simulation , Nucleotides/chemistry , Nucleotides/metabolism , Thermodynamics , Water/chemistry , Water/metabolismABSTRACT
We have developed Differential Specificity and Energy Landscape (DiSEL) analysis to comprehensively compare DNA-protein interactomes (DPIs) obtained by high-throughput experimental platforms and cutting edge computational methods. While high-affinity DNA binding sites are identified by most methods, DiSEL uncovered nuanced sequence preferences displayed by homologous transcription factors. Pairwise analysis of 726 DPIs uncovered homolog-specific differences at moderate- to low-affinity binding sites (submaximal sites). DiSEL analysis of variants of 41 transcription factors revealed that many disease-causing mutations result in allele-specific changes in binding site preferences. We focused on a set of highly homologous factors that have different biological roles but "read" DNA using identical amino acid side chains. Rather than direct readout, our results indicate that DNA noncontacting side chains allosterically contribute to sculpt distinct sequence preferences among closely related members of transcription factor families.
Subject(s)
DNA/metabolism , Transcription Factors/metabolism , Binding Sites , SELEX Aptamer Technique , ThermodynamicsABSTRACT
Post-transcriptional modification of snRNA is central to spliceosome function. Usb1 is an exoribonuclease that shortens the oligo-uridine tail of U6 snRNA, resulting in a terminal 2',3' cyclic phosphate group in most eukaryotes, including humans. Loss of function mutations in human Usb1 cause the rare disorder poikiloderma with neutropenia (PN), and result in U6 snRNAs with elongated 3' ends that are aberrantly adenylated. Here, we show that human Usb1 removes 3' adenosines with 20-fold greater efficiency than uridines, which explains the presence of adenylated U6 snRNAs in cells lacking Usb1. We determined three high-resolution co-crystal structures of Usb1: wild-type Usb1 bound to the substrate analog adenosine 5'-monophosphate, and an inactive mutant bound to RNAs with a 3' terminal adenosine and uridine. These structures, along with QM/MM MD simulations of the catalytic mechanism, illuminate the molecular basis for preferential deadenylation of U6 snRNA. The extent of Usb1 processing is influenced by the secondary structure of U6 snRNA.
Subject(s)
Adenosine/metabolism , Phosphoric Diester Hydrolases/metabolism , RNA, Small Nuclear/metabolism , Uridine/metabolism , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Base Sequence , Crystallography, X-Ray , Genetic Predisposition to Disease/genetics , Humans , Models, Molecular , Mutation , Neutropenia/genetics , Neutropenia/metabolism , Nucleic Acid Conformation , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Protein Binding , Protein Conformation , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/genetics , Skin Abnormalities/genetics , Skin Abnormalities/metabolism , Substrate SpecificityABSTRACT
We discuss the application of quantum mechanics/molecular mechanics (QM/MM) free energy simulations to the analysis of phosphoryl transfers catalyzed by two enzymes: alkaline phosphatase and myosin. We focus on the nature of the transition state and the issue of mechanochemical coupling, respectively, in the two enzymes. The results illustrate unique insights that emerged from the QM/MM simulations, especially concerning the interpretation of experimental data regarding the nature of enzymatic transition states and coupling between global structural transition and catalysis in the active site. We also highlight a number of technical issues worthy of attention when applying QM/MM free energy simulations, and comment on a number of technical and mechanistic issues that require further studies.
Subject(s)
Alkaline Phosphatase/metabolism , Molecular Dynamics Simulation , Myosins/metabolism , Alkaline Phosphatase/chemistry , Myosins/chemistry , Quantum TheoryABSTRACT
The fusion pore is the first crucial intermediate formed during exocytosis, yet little is known about the mechanisms that determine the size and kinetic properties of these transient structures. Here, we reduced the number of available SNAREs (proteins that mediate vesicle fusion) in neurons and observed changes in transmitter release that are suggestive of alterations in fusion pores. To investigate these changes, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores and enabled the escape of larger cargoes. To determine whether this effect was due to changes in nascent pore size or to changes in stability, we developed an approach that uses nanodiscs and planar lipid bilayer electrophysiology to afford microsecond resolution at the single event level. Both pore size and stability were affected by SNARE copy number. Increasing the number of vesicle (v)-SNAREs per nanodisc from three to five caused a twofold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, pairing of v-SNAREs and target (t)-SNAREs to form trans-SNARE complexes was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, which mimics the action of botulinum neurotoxin A, markedly reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determines fundamental properties of individual pores.
Subject(s)
Cell Membrane/metabolism , Exocytosis , Membrane Fusion , Porosity , SNARE Proteins/metabolism , Animals , Botulinum Toxins, Type A/metabolism , Excitatory Postsynaptic Potentials , Lipid Bilayers/metabolism , Mice , Neurons/cytology , Neurons/metabolism , Neurotransmitter Agents/metabolism , Rats , Rats, Sprague-Dawley , Secretory Vesicles/metabolismABSTRACT
The mechanism of ATP hydrolysis in the myosin motor domain is analyzed using a combination of DFTB3/CHARMM simulations and enhanced sampling techniques. The motor domain is modeled in the pre-powerstroke state, in the post-rigor state, and as a hybrid based on the post-rigor state with a closed nucleotide-binding pocket. The ATP hydrolysis activity is found to depend on the positioning of nearby water molecules, and a network of polar residues facilitates proton transfer and charge redistribution during hydrolysis. Comparison of the observed hydrolysis pathways and the corresponding free energy profiles leads to detailed models for the mechanism of ATP hydrolysis in the pre-powerstroke state and proposes factors that regulate the hydrolysis activity in different conformational states. In the pre-powerstroke state, the scissile Pγ-O3ß bond breaks early in the reaction. Proton transfer from the lytic water to the γ-phosphate through active site residues is an important part of the kinetic bottleneck; several hydrolysis pathways that feature distinct proton transfer routes are found to have similar free energy barriers, suggesting a significant degree of plasticity in the hydrolysis mechanism. Comparison of hydrolysis in the pre-powerstroke state and the closed post-rigor model suggests that optimization of residues beyond the active site for electrostatic stabilization and preorganization is likely important to enzyme design.
Subject(s)
Adenosine Triphosphate/chemistry , Myosin Type II/chemistry , Protons , Protozoan Proteins/chemistry , Water/chemistry , Adenosine Triphosphate/metabolism , Biocatalysis , Biomechanical Phenomena , Catalytic Domain , Dictyostelium/chemistry , Dictyostelium/enzymology , Dictyostelium/genetics , Gene Expression , Hydrolysis , Kinetics , Models, Molecular , Myosin Type II/genetics , Myosin Type II/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Static Electricity , Thermodynamics , Water/metabolismABSTRACT
Enzymes are powerful catalysts, and a thorough understanding of the sources of their catalytic power will facilitate many medical and industrial applications. Here we have studied the catalytic mechanism of alkaline phosphatase (AP), which is one of the most catalytically proficient enzymes known. We have used quantum mechanics calculations and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to model a variety of isotope effects relevant to the reaction of AP. We have calculated equilibrium isotope effects (EIEs), binding isotope effects (BIEs), and kinetic isotope effects (KIEs) for a range of phosphate mono- and diester substrates. The results agree well with experimental values, but the model for the reaction's transition state (TS) differs from the original interpretation of those experiments. Our model indicates that isotope effects on binding make important contributions to measured KIEs on V/K, which complicated interpretation of the measured values. Our results provide a detailed interpretation of the measured isotope effects and make predictions that can test the proposed model. The model indicates that the substrate is deformed in the ground state (GS) of the reaction and partially resembles the TS. The highly preorganized active site preferentially binds conformations that resemble the TS and not the GS, which induces the substrate to adapt to the enzyme, rather than the other way around-as with classic "induced fit" models. The preferential stabilization of the TS over the GS is what lowers the barrier to the chemical step.
Subject(s)
Alkaline Phosphatase/metabolism , Oxygen Isotopes , Alkaline Phosphatase/chemistry , Catalytic Domain , Kinetics , Models, Molecular , Protein BindingABSTRACT
A reaction's transition state (TS) structure plays a critical role in determining reactivity and has important implications for the design of catalysts, drugs, and other applications. Here, we explore TS structure in the enzyme alkaline phosphatase using hybrid Quantum Mechanics/Molecular Mechanics simulations. We find that minor perturbations to the substrate have major effects on TS structure and the way the enzyme stabilizes the TS. Substrates with good leaving groups (LGs) have little cleavage of the phosphorus-LG bond at the TS, while substrates with poor LGs have substantial cleavage of that bond. The results predict nonlinear free energy relationships for a single rate-determining step, and substantial differences in kinetic isotope effects for different substrates; both trends were observed in previous experimental studies, although the original interpretations differed from the present model. Moreover, due to different degrees of phosphorus-LG bond cleavage at the TS for different substrates, the LG is stabilized by different interactions at the TS: while a poor LG is directly stabilized by an active site zinc ion, a good LG is mainly stabilized by active site water molecules. Our results demonstrate the considerable plasticity of TS structure and stabilization in enzymes. Furthermore, perturbations to reactivity that probe TS structure experimentally (i.e., substituent effects) may substantially perturb the TS they aim to probe, and thus classical experimental approaches such as free energy relations should be interpreted with care.
Subject(s)
Alkaline Phosphatase/chemistry , Escherichia coli/enzymology , Models, Theoretical , Binding Sites , Catalysis , Computer Simulation , Hydrolysis , Phase Transition , Protein Conformation , Substrate SpecificityABSTRACT
We discuss the description of water and hydration effects that employs an approximate density functional theory, DFTB3, in either a full QM or QM/MM framework. The goal is to explore, with the current formulation of DFTB3, the performance of this method for treating water in different chemical environments, the magnitude and nature of changes required to improve its performance, and factors that dictate its applicability to reactions in the condensed phase in a QM/MM framework. A relatively minor change (on the scale of kBT) in the O-H repulsive potential is observed to substantially improve the structural properties of bulk water under ambient conditions; modest improvements are also seen in dynamic properties of bulk water. This simple change also improves the description of protonated water clusters, a solvated proton, and to a more limited degree, a solvated hydroxide. By comparing results from DFTB3 models that differ in the description of water, we confirm that proton transfer energetics are adequately described by the standard DFTB3/3OB model for meaningful mechanistic analyses. For QM/MM applications, a robust parametrization of QM-MM interactions requires an explicit consideration of condensed phase properties, for which an efficient sampling technique was developed recently and is reviewed here. The discussions help make clear the value and limitations of DFTB3 based simulations, as well as the developments needed to further improve the accuracy and transferability of the methodology.
Subject(s)
Models, Molecular , Water/chemistry , Quantum TheoryABSTRACT
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.
Subject(s)
Hydrogen/metabolism , Mutation , Tetrahydrofolate Dehydrogenase/genetics , Molecular Dynamics Simulation , Quantum TheoryABSTRACT
We recently reported abnormal secondary deuterium kinetic isotope effects (2° KIEs) for hydride transfer reactions from alcohols to carbocations in acetonitrile (Chem. Comm. 2012, 48, 11337). Experimental 2° KIE values were found to be inflated on the 9-C position in the xanthylium cation but deflated on the ß-C position in 2-propanol with respect to the values predicted by the semi-classical transition-state theory. No primary (1°) isotope effect on 2° KIEs was observed. Herein, the KIEs were replicated by the Marcus-like H-tunneling model that requires a longer donor-acceptor distance (DAD) in a lighter isotope transfer process. The 2° KIEs for a range of potential tunneling-ready-states (TRSs) of different DADs were calculated and fitted to the experiments to find the TRS structure. The observed no effect of 1° isotope on 2° KIEs is explained in terms of the less sterically hindered TRS structure so that the change in DAD due to the change in 1° isotope does not significantly affect the reorganization of the 2° isotope and hence the 2° KIE. The effect of 1° isotope on 2° KIEs may be expected to be more pronounced and thus observable in reactions occurring in restrictive environments such as the crowded and relatively rigid active site of enzymes.
Subject(s)
Alcohols/chemistry , Deuterium/chemistry , Isotopes/chemistry , Acetonitriles/chemistry , Catalytic Domain , Computers, Molecular , Kinetics , Models, Chemical , SolutionsABSTRACT
Isotopically labeled enzymatic substrates and biological metabolites are useful for many mechanistic analyses, particularly the study of kinetic and equilibrium isotope effects, determining the stereospecificity of enzymes, and resolving metabolic pathways. Here, we present the one-pot synthesis, purification, and kinetic analysis of 7R-[(2) H]-phenyl-[(14) C]-benzyl alcohol. The procedure involves a chemoenzymatic synthesis that couples formate dehydrogenase to alcohol dehydrogenase with a catalytic amount of nicotinamide cofactor. The reaction goes to completion overnight, and the measurement of a competitive kinetic isotope effect on the enzymatic oxidation of the purified product identified no (1) H contamination. This measurement is very sensitive to such isotopic contamination and verified the high level of isotopic and enantiomeric purity yielded by the new synthetic procedure.
Subject(s)
Benzyl Alcohol/chemistry , Benzyl Alcohol/metabolism , Benzyl Alcohols/chemistry , Isotope Labeling , Alcohol Dehydrogenase/chemistry , Carbon Radioisotopes/chemistry , Deuterium , Formate Dehydrogenases/chemistry , Kinetics , Niacinamide/chemistry , StereoisomerismABSTRACT
Enzymes use a number of common cofactors as sources of hydrogen to drive biological processes, but the physics of the hydrogen transfers to and from these cofactors is not fully understood. Researchers study the mechanistically important contributions from quantum tunneling and enzyme dynamics and connect those processes to the catalytic power of enzymes that use these cofactors. Here we describe some progress that has been made in studying these reactions, particularly through the use of kinetic isotope effects (KIEs). We first discuss the general theoretical framework necessary to interpret experimental KIEs, and then describe practical uses for KIEs in the context of two case studies. The first example is alcohol dehydrogenase, which uses a nicotinamide cofactor to catalyze a hydride transfer, and the second example is thymidylate synthase, which uses a folate cofactor to catalyze both a hydride and a proton transfer.
Subject(s)
Coenzymes/metabolism , Hydrogen/metabolism , Alcohol Dehydrogenase/metabolism , Animals , Folic Acid/metabolism , Humans , Kinetics , Models, Molecular , Niacinamide/metabolism , Thermodynamics , Thymidylate Synthase/metabolismABSTRACT
The physical mechanism of C-H bond activation by enzymes is the subject of intense study, and we have tested the predictions of two competing models for C-H activation in the context of alcohol dehydrogenase. The kinetic isotope effects (KIEs) in this enzyme have previously suggested a model of quantum mechanical tunneling and coupled motion of primary (1°) and secondary (2°) hydrogens. Here we measure the 2° H/T KIEs with both H and D at the 1° position and find that the 2° KIE is significantly deflated with D-transfer, consistent with the predictions of recent Marcus-like models of H-transfer. The results suggest that the fast dynamics of H-tunneling result in a 1° isotope effect on the structure of the tunneling ready state: the trajectory of D-transfer goes through a shorter donor-acceptor distance than that of H-transfer.
Subject(s)
Alcohol Dehydrogenase/chemistry , Benzyl Alcohol/chemistry , Models, Molecular , Alcohol Dehydrogenase/metabolism , Oxidation-ReductionABSTRACT
Kinetic Isotope effects (KIEs) have long served as a probe for the mechanisms of both enzymatic and solution reactions. Here, we discuss various models for the physical sources of KIEs, how experimentalists can use those models to interpret their data, and how the focus of traditional models has grown to a model that includes motion of the enzyme and quantum mechanical nuclear tunneling. We then present two case studies of enzymes, thymidylate synthase and alcohol dehydrogenase, and discuss how KIEs have shed light on the C-H bond cleavages those enzymes catalyze. We will show how the combination of both experimental and computational studies has changed our notion of how these enzymes exert their catalytic powers.
Subject(s)
Alcohol Dehydrogenase/chemistry , Computer Simulation , Models, Chemical , Nucleoside-Phosphate Kinase/chemistry , Isotopes/chemistry , KineticsABSTRACT
Kinetic isotope effects (KIEs) and their temperature dependence can probe the structural and dynamic nature of enzyme-catalyzed proton or hydride transfers. The molecular interpretation of their temperature dependence requires expensive and specialized quantum mechanics/molecular mechanics (QM/MM) calculations to provide a quantitative molecular understanding. Currently available phenomenological models use a nonadiabatic assumption that is not appropriate for most hydride and proton-transfer reactions, while others require more parameters than the experimental data justify. Here we propose a phenomenological interpretation of KIEs based on a simple method to quantitatively link the size and temperature dependence of KIEs to a conformational distribution of the catalyzed reaction. This model assumes adiabatic hydrogen tunneling, and by fitting experimental KIE data, the model yields a population distribution for fluctuations of the distance between donor and acceptor atoms. Fits to data from a variety of proton and hydride transfers catalyzed by enzymes and their mutants, as well as nonenzymatic reactions, reveal that steeply temperature-dependent KIEs indicate the presence of at least two distinct conformational populations, each with different kinetic behaviors. We present the results of these calculations for several published cases and discuss how the predictions of the calculations might be experimentally tested. This analysis does not replace molecular QM/MM investigations, but it provides a fast and accessible way to quantitatively interpret KIEs in the context of a Marcus-like model.
Subject(s)
Enzymes/chemistry , Hydrogen/chemistry , Animals , Humans , Kinetics , Models, Molecular , Temperature , ThermodynamicsABSTRACT
The mechanisms of enzyme-catalyzed reactions are medicinally important and present a fascinating intellectual challenge. Many experimental and theoretical techniques can shed light on these mechanisms, and here, we shall focus on the utility of kinetic isotope effects (KIEs) to study enzymatic reactions that involve hydrogen transfers. We will provide a short background on the prevailing models to interpret KIEs and then present more detailed reviews of two model enzymes: alcohol dehydrogenase and thymidylate synthase. These two examples provide a context to describe the types of experiments and theoretical calculations that drive this field forward and the kind of information each can furnish. We emphasize the importance of cooperation between experimentalists and theoreticians to continue the progress toward a comprehensive theory of enzyme catalysis.
Subject(s)
Alcohol Dehydrogenase/metabolism , Hydrogen/metabolism , Thymidylate Synthase/metabolism , Alcohol Dehydrogenase/chemistry , Biocatalysis , Hydrogen/chemistry , Thymidylate Synthase/chemistry , Thymidylate Synthase/geneticsABSTRACT
For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (> 1) 2 degrees kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (< 1), this KIE eludes the traditional interpretation of 2 degrees KIEs. It does, however, enable the development of a comprehensive model for the "tunneling ready state" (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate.