Universal Glycosyltransferase Continuous Assay for Uniform Kinetics and Inhibition Database Development and Mechanistic Studies Illustrated on ST3GAL1, C1GALT1, and FUT1

Chemical systems glycobiology requires experimental and computational tools to make possible big data analytics benefiting genomics and proteomics. The impediment to tool development is that the nature of glycan construction and mutation is not template driven but rests on cooperative glycosyltransferase (GT) catalytic synthesis. What is needed is the collation of kinetics and inhibition data in a standardized form to make possible analytics of glycan and glycoconjugate synthesis, mechanism extraction, and pattern recognition. Currently, kinetics assays in use for GTs are not universal in processing nucleoside phosphate UDP, GDP, and CMP donor-based glycosylation reactions due to limitations in accuracy and large substrate volume requirements. Here we present a universal glycosyltransferase continuous (UGC) assay able to measure the declining concentration of the NADH reporter molecule through fluorescence spectrophotometry and, therefore, determine reaction rate parameters. The development and parametrization of the assay is based on coupling the nucleotide released from GT reactions with pyruvate kinase, via nucleoside diphosphate kinase (NDK) in the case of NDP-based donor reactions. In the case of CMP-based reactions, the coupling is carried out via another kinase, cytidylate kinase in combination with NDK, which phosphorylates CMP to CDP, then CDP to CTP. Following this, we conduct kinetics and inhibition assay studies on the UDP, GDP, and CMP-based glycosylation reactions, specifically C1GAlT1, FUT1, and ST3GAL1, to represent each class of donor, respectively. The accuracy of calculating initial rates using the continuous assay compared to end point (noncontinuous) assays is demonstrated for the three classes of GTs. The previously identified natural product soyasaponin1 inhibitor was used as a model to demonstrate the application of the UGC assay as a standardized inhibition assay for GTs. We show that the dose response of ST3GAL1 to a serial dilution of Soyasaponin1 has time-dependent inhibition. This brings into question previous inhibition findings, arrived at using an end point assay, that have selected a seemingly random time point to measure inhibition. Consequently, using standardized Km values taken from the UGC assay study, ST3GAL1 was shown to be the most responsive enzyme to soyasaponin1 inhibition, followed by FUT1, then C1GALT1 with IC50 values of 37, 52, and 886 μM respectivel

Profiling Transition-State Configurations on the <i>Trypanosoma cruzi</i> <i>trans</i>-Sialidase Free-Energy Reaction Surfaces

Enzymatically catalyzed reactions pass from reactants to products via transition states that are short-lived and potentially characterized from free-energy reaction surfaces. We compute the reaction surface for <i>Trypanosoma cruzi trans</i>-sialidase using the Free Energy from Adaptive Reaction Coordinate Forces method. The reaction coordinates are the bonds between the sialic acid and the leaving group (TYR₃₄₂) and the sialic acid and the nucpleophile (ASP₅₉). We are able to track progress of the reaction trajectories up to (incomplete), about (recrossed), and across (crossed) the col that divides the reactant (covalent intermediate) and product (Michaelis complex) surfaces. More than 40 transition state configurations were isolated from these trajectories, and the sialic acid substrate conformations were analyzed as well as the substrate interactions with the nucleophile and catalytic acid/base. A successful barrier crossing requires that the substrate passes through a family of E₅, ⁴H₅, and ⁶H₅ pucker conformations. These puckers interact slightly differently with the enzyme. The E₅ and ⁴H₅ conformations have a high-frequency hydrogen bonding with Asp₉₆, while ⁶H₅ puckers show increased hydrogen bonding between sialic acid O-8–Glu₂₃₀. Our analysis of <i>Trypanosoma cruzi trans</i>-sialidase configurations that populate the col separating the reactant from product surfaces brings new evidence to the prevailing premise that there are several pathways from reactant to product passing through the saddle and successful product formation is not restricted to the minimum energy path and transition state

Multidimensional Reaction Dynamics Reveal How the Enzyme TcTS Suppresses Competing Side Reactions and Their Side Products

The suppression of competing reactions that lead to side products is one of the key mechanistic actions defining enzyme catalysis. The transfer of sialic acid (SA) in a water solution is susceptible to two competing side reactions, but a single product is the outcome in the glycosylation and deglycosylation steps of the <i>trans</i>-sialidase (TS) in the Trypanosoma cruzi (T. cruzi) parasite, generally known as TcTS. We use multidimensional QM/MM free energy computations to reveal a competition between a minor elimination reaction and the dominant displacement reaction present in both steps. The simultaneous monitoring of the progression of the competing reactions reveals lower barriers in the free energy profiles, a greater sampling of favorable reactant stereoelectronic alignments, and a greater number of possible transition paths leading to successful crossing reaction trajectories for the dominant displacement reactions in comparison with those of the elimination reactions

Evaluating AM1/d-CB1 for Chemical Glycobiology QM/MM Simulations

The newly parametrized AM1/d-CB1 is evaluated for its performance in modeling monosaccharide structure, carbohydrate ring pucker, amino acid proton transfer, DNA base pair interactions, carbohydrate–aromatic π interactions, and phosphates that are prominent in glycosyltransferases. The accuracy of the method in these computations is compared to a comprehensive range of NDDO methods commonly used to study glycan structure and reactivity in chemical biology. AM1/d-CB1 shows significant improvement over existing NDDO type methods in the computation of five and six membered carbohydrate ring pucker free energy surfaces. Moreover, the computation of carbohydrate amino acid interactions commonly present in catalytic domains and binding sites are improved over existing NDDO methods. AM1/d-CB1 shows slight improvement for carbohydrate−aromatic π interactions compared to a commonly used NDDO method (AM1). The method is applied to a glycosyltransferase reaction, where it is the only NDDO method able to achieve an optimized reaction profile. Moreover, a comparison of the geometry optimized computations of the reaction scheme give a transition state energy barrier that best compares with DFT (MPW1K). Overall, AM1/d-CB1 is shown to significantly improve on existing NDDO methods in modeling chemical glycobiological events

Simple Link Atom Saccharide Hybrid (SLASH) Treatment for Glycosidic Bonds at the QM/MM Boundary

We investigated link atom approaches for treating the polar C–O bond with particular reference to the glycosidic bond found in complex carbohydrates. We show that cutting this bond after the oxygen in the QM region and saturating the QM system with a hydrogen link atom leads to greater conformational and configurational accuracy at the boundary compared with cutting the bond before oxygen and saturating the QM system with a halogen link atom to represent the oxygen. Furthermore, we find that balancing the MM atom charges and redistributing the boundary atom charges at the QM/MM boundary minimizes the effect of the link atom, both energetically and structurally. This is illustrated via a series of calculations on a set of carbohydrate and carbohydrate-like model compounds. Finally, we confirm the validity of our model by performing molecular dynamics simulations for a typical disaccharide model compound in water. Our postsimulation conformational and configurational analyses show that the oxygen-to-water hydrogen pair distribution functions and the Φ,Ψ distributions at the glycosidic boundary between the quantum and classical regions compare favorably with results obtained from complete QM and complete MM treatments of the saccharide

Computational Rationale for the Selective Inhibition of the Herpes Simplex Virus Type 1 Uracil-DNA Glycosylase Enzyme

The herpes simplex virus uracil-DNA glycosylase (hsvUNG) enzyme is responsible for the reactivation of the virus from latency and efficient viral replication in nerve tissue. The lack of uracil-DNA glycosylase enzyme in human neurons and the continuous deamination of cytosine create an environment where the presence of viral uracil-DNA glycosylase is a necessity for the proliferation of the virus. A series of 6-(4-alkylanilino)-uracil inhibitors has been developed that selectively and strongly binds to the hsvUNG enzyme while weakly binding to human uracil-DNA glycosylase (hUNG). Here, by using a combination of sequence and structural comparisons between the two enzymes along with free energy of binding computations and principal component analysis of the ligands, we investigate and rationalize the inhibitory effect of the 6-(4-alkylanilino)-uracil series as a function of alkyl chain length on the hsvUNG. The results of these computations corroborate the experimental finding that the inhibitor with an octyl aliphatic chain selectively binds hsvUNG best. More importantly we find that 6-(4-octylanilino)-uracil’s selective inhibition of hsvUNG over hUNG is due to the combination of the solution preconfigured bent conformation of that specific chain length and the position of HIS92 (absent in hUNG) just outside hsvUNG’s hydrophobic gorge lying adjacent to its uracil binding pocket. The similarities between the uracil binding pockets in hsvUNG and hUNG obfuscate an understanding of the preferential inhibition of the virus enzyme. However, the differences in the enzymes’ shallow hydrophobic grooves adjacent to the binding pockets, such as the gorge we identify here, rationalizes 6-(4-alkylanilino)-uracil with an octyl chain length as an excellent pharmacophore template for hsvUNG inhibitor design

AM1/d-CB1: A Semiempirical Model for QM/MM Simulations of Chemical Glycobiology Systems

A semiempirical method based on the AM1/d Hamiltonian is introduced to model chemical glycobiological systems. We included in the parameter training set glycans and the chemical environment often found about them in glycoenzymes. Starting with RM1 and AM1/d-PhoT models we optimized H, C, N, O, and P atomic parameters targeting the best performing molecular properties that contribute to enzyme catalyzed glycan reaction mechanisms. The training set comprising glycans, amino acids, phosphates and small organic model systems was used to derive parameters that reproduce experimental data or high-level density functional results for carbohydrate, phosphate and amino acid heats of formation, amino acid proton affinities, amino acid and monosaccharide dipole moments, amino acid ionization potentials, water-phosphate interaction energies, and carbohydrate ring pucker relaxation times. The result is the AM1/d-Chemical Biology 1 or AM1/d-CB1 model that is considerably more accurate than existing NDDO methods modeling carbohydrates and the amino acids often present in the catalytic domains of glycoenzymes as well as the binding sites of lectins. Moreover, AM1/d-CB1 computed proton affinities, dipole moments, ionization potentials and heats of formation for transition state puckered carbohydrate ring conformations, observed along glycoenzyme catalyzed reaction paths, are close to values computed using DFT M06-2X. AM1/d-CB1 provides a platform from which to accurately model reactions important in chemical glycobiology

Molecular Structures and Solvation of Free Monomeric and Dimeric Ferriheme in Aqueous Solution: Insights from Molecular Dynamics Simulations and Extended X‑ray Absorption Fine Structure Spectroscopy

CHARMM force field parameters have been developed to model nonprotein bound five-coordinate ferriheme (ferriprotoporphyrin IX) species in aqueous solution. Structures and solvation were determined from molecular dynamics (MD) simulations at 298 K of monomeric [HO-ferriheme]^2–, [H₂O-ferriheme]⁻, and [H₂O-ferriheme]⁰; π–π dimeric [(HO-ferriheme)₂]^4–, [(H₂O-ferriheme)­(HO-ferriheme)]^3–, [(H₂O-ferriheme)₂]^2–, and [(H₂O-ferriheme)₂]⁰; and μ-oxo dimeric [μ-(ferriheme)₂O]^4–. Solvation of monomeric species predominated around the axial ligand, meso-hydrogen atoms of the porphyrin ring (H_meso), and the unligated face. Existence of π–π ferriheme dimers in aqueous solution was supported by MD calculations where such dimers remained associated over the course of the simulation. Porphyrin rings were essentially coplanar. In these dimers major and minor solvation was observed around the axial ligand and H_meso positions, respectively. In μ-oxo ferriheme, strong solvation of the unligated face and bridging oxide ligand was observed. The solution structure of the μ-oxo dimer was investigated using extended X-ray absorption fine structure (EXAFS) spectroscopy. The EXAFS spectrum obtained from frozen solution was markedly different from that recorded on dried μ-oxo ferriheme solid. Inclusion of five solvent molecules obtained from spatial distribution functions in the structure generated from MD simulation was required to produce acceptable fits to the EXAFS spectra of the dimer in solution, while the solid was suitably fitted using the crystal structure of μ-oxo ferriheme dimethyl ester which included no solvent molecules