Alexandra T. P. Carvalho

The First Mammalian Aldehyde Oxidase Crystal Structure: Insights Into Substrate specificity

Background: Aldehyde oxidases have pharmacological relevance, and AOX3 is the major drug-metabolizing enzyme in rodents. Results: The crystal structure of mouse AOX3 with kinetics and molecular docking studies provides insights into its enzymatic characteristics. Conclusion: Differences in substrate and inhibitor specificities can be rationalized by comparing the AOX3 and xanthine oxidase structures. Significance: The first aldehyde oxidase structure represents a major advance for drug design and mechanistic studies. Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity.

Determination of the ΔpKa between the active site cysteines of thioredoxin and DsbA

Thioredoxin superfamily members share a considerable degree of structural similarity, with a conserved CXiXjC motif at the active site, where C stand for two cysteines that alternate between a reduced thiol and oxidized disulfide states, and Xiand Xj are two amino acids different in each family member. Despite these similarities, they display very different redox potentials and pKas for the active site dithiol, and fulfill different physiological roles. Thioredoxin, for example, promotes the reduction of disulfide bonds, while DsbA promotes their oxidation in prokaryotic cells. The factors that promote these differences are still not fully understood. However, it is generally accepted that the different stabilities of the redox active disulfide bond depends on the degree of stabilization, in the reduced state, of the thiolate of one of the active site cysteines (nucleophilic cysteine). In this work we have used QM/MM methods to compare and characterize the active site dithiols of both enzymes, and to shed some light on the structural features responsible for the large differences in pKa and redox potential between two homologous enzymes, thioredoxin and DsbA. We have also analyzed the main factors pointed out in the literature as responsible for their different properties. We obtained the value of 4.5 for pKa difference (ΔpKa) between the nucleophilic cysteines of both enzymes, which is in excellent agreement with most of the experimental values. Additionally, we found that the principal differentiating factor responsible for this observed ΔpKa are the α2‐alpha helices, which greatly contribute to the mentioned value, by stabilizing the DsbA thiolate in a much greater extend than the thioredoxin thiolate. A double mutation of the conserved residues Asp26 and Lys57, in thioredoxin, and Glu24 Lys58, in DsbA, by alanines did not change the ΔpKa value; this supports the hypothesis that these residues are not involved in the differentiation of the properties of the active centre dithiol. However, we found out that these residues are important for the stabilization of the nucleophilic thiolate. The Xi and Xj residues also do not seem to promote the stabilization of the thiolates. In fact, the corresponding double alanine mutants are more stable than the wild‐type enzymes. However, these residues are involved in the differentiation between thioredoxin and DsbA, stabilizing the DsbA thiolate by a larger extent than the thioredoxin thiolate.

Parameters for molecular dynamics simulations of iron-sulfur proteins.

Iron‐sulfur proteins involved in electron transfer reactions have finely tuned redox potentials, which allow them to be highly efficient and specific. Factors such as metal center solvent exposure, interaction with charged residues, or hydrogen bonds between the ligand residues and amide backbone groups have all been pointed out to cause such specific redox potentials. Here, we derived parameters compatible with the AMBER force field for the metal centers of iron‐sulfur proteins and applied them in the molecular dynamics simulations of three iron‐sulfur proteins. We used density‐functional theory (DFT) calculations and Seminarios method for the parameterization. Parameter validation was obtained by matching structures and normal frequencies at the quantum mechanics and molecular mechanics levels of theory. Having guaranteed a correct representation of the protein coordination spheres, the amide H‐bonds and the water exposure to the ligands were analyzed. Our results for the pattern of interactions with the metal centers are consistent to those obtained by nuclear magnetic resonance spectroscopy (NMR) experiments and DFT calculations, allowing the application of molecular dynamics to the study of those proteins.

Role of the variable active site residues in the function of thioredoxin family oxidoreductases.

The enzymes of the thioredoxin family fulfill a wide range of physiological functions. Although they possess a similar CXYC active site motif, with identical environment and stereochemical properties, the redox potential and pKa of the cysteine pair varies widely across the family. As a consequence, each family member promotes oxidation or reduction reactions, or even isomerization reactions. The analysis of the three‐dimensional structures gives no clues to identify the molecular source for the different active site properties. Therefore, we carried out a set of quantum mechanical calculations in active site models to gain more understanding on the elusive molecular‐level origin of the differentiation of the properties across the family. The obtained results, together with earlier quantum mechanical calculations performed in our laboratories, gave rise to a consistent line of evidence, which points to the fact that both active site cysteines play an important role in the differentiation. In contrary to what was assumed, differentiation is not achieved through a different stabilization of the solvent exposed cysteine but, instead, through a fine tuning of the nucleophilicity of both active site cysteines. Reductant enzymes have both cysteine thiolates poorly stabilized, oxidant proteins have both cysteine thiolates highly stabilized, and isomerases have one thiolate (solvent exposed) poorly stabilized and the other (buried) thiolate highly stabilized. The feasibility of shifting the chemical equilibrium toward oxidation, reduction, or isomerization only through subtle electrostatic effects is quite unusual, and it relies on the inherent thermoneutrality of the catalytic steps carried out by a set of chemically equivalent entities all of which are cysteine thiolates. Such pattern of stabilization/destabilization, detected in our calculations is fully consistent with the observed physiological roles of this family of enzymes.

New designs for MRI contrast agents.

New designs for Magnetic Resonance Imaging contrast agents are presented. Essentially, they all are host–guest inclusion complexes between γ-cyclodextrins and polyazamacrocycles of gadolinium (III) ion. Substitutions have been made to the host to optimise the host–guest association. Molecular mechanics calculations have been performed, using the UFF force field for metals, to decide on the suitability of the substitutions, and to evaluate the host–guest energies of association. Interesting general conclusions have been obtained, concerning the improvement of Magnetic Resonance Imaging contrast agents; namely, a set of rational methodologies have been deduced to improve the association between the gadolinium (III) chelates and the cyclodextrins, and their efficiency is demonstrated with a large set of substituted complexes, opening new doors to increase the diagnostic capabilities of Magnetic Resonance Imaging.

Electronic structure investigation and parametrization of biologically relevant iron-sulfur clusters.

The application of classical molecular dynamics simulations to the study of metalloenzymes has been hampered by the lack of suitable molecular mechanics force field parameters to treat the metal centers within standard biomolecular simulation packages. These parameters cannot be generalized, nor be easily automated, and hence should be obtained for each system separately. Here we present density functional theory calculations on [Fe2S2(SCH3)4]2+/+, [Fe3S4(SCH3)3]+/0 and [Fe4S4(SCH3)4]2+/+ and the derivation of parameters that are compatible with the AMBER force field. Molecular dynamics simulations performed using these parameters on respiratory Complex II of the electron transport chain showed that the reduced clusters are more stabilized by the protein environment, which leads to smaller changes in bond lengths and angles upon reduction. This effect is larger in the smaller iron-sulfur cluster, [Fe2S2(SCH3)4]2+/+.

Molecular Insights into the Mechanisms of HIV-1 Reverse Transcriptase Resistance to Nucleoside Analogs

The causative agent of acquired immunodeficiency syndrome, HIV-1, depends on one of its enzymes, reverse transcriptase, to copy its single stranded RNA genome into a double stranded DNA nucleic acid suitable for integration in the host cell genome. In the last two decades, the advances in the knowledge of the kinetic mechanism of reverse transcription and in the determination of the crystallographic structures for the complexes of the enzyme with substrates and products were huge. However, all of this knowledge resulted in the design of RT inhibitors for which the virus, after a short period of exposure, becomes less susceptible, due to the development of resistance. The development of resistance is caused by the high frequency of viral mutation and the toxicity of those same drugs. Therefore, a closer look at all the available information might shed some light into this subject and help to develop new strategies to overcome the lack of long term clinical efficiency of these drugs. Here, we present a critical atomic level study of all the mutations that have been detected and reported so far, as a reaction of the enzyme to counteract the action of the inhibitors.

Molecular Dynamics Model of Unliganded HIV-1 Reverse Transcriptase

HIV-1 RT is one of the most important antiviral targets in the treatment of acquired immunodeficiency syndrome (AIDS). Several crystallographic structures are available for this enzyme, mostly with bound inhibitors. Despite their importance for structure based drug design towards new anti-HIV retrovirals, the X-ray structures of the unliganded enzyme could only be obtained incomplete, with a low resolution and until recently even the conformation of the p66 thumb was controversial. In this work we have aligned different X-ray RT structures, and built up a computational model of RT using homology modeling, which was afterwards refined and validated through MD simulations with explicit solvent. The model enzyme was structurally stable through the whole MD simulation, showing a RMSD of 2 Angstrom from the starting geometry. The Ramanchandram plot has improved along the simulation. Both intra-domain and interdomain movements were observed. The thumb kept its closed conformation through the whole simulation. A contact map, hydration sites study and a detailed analysis of the solvation of the nucleotide binding site are also presented.

Why the Flavin Adenine Dinucleotide (FAD) Cofactor Needs To Be Covalently Linked to Complex II of the Electron‐Transport Chain for the Conversion of FADH2 into FAD

Abstract A covalently bound flavin cofactor is predominant in the succinate‐ubiquinone oxidoreductase (SQR; Complex II), an essential component of aerobic electron transport, and in the menaquinol‐fumarate oxidoreductase (QFR), the anaerobic counterpart, although it is only present in approximately 10 % of the known flavoenzymes. This work investigates the role of this 8α‐N3‐histidyl linkage between the flavin adenine dinucleotide (FAD) cofactor and the respiratory Complex II. After parameterization with DFT calculations, classical molecular‐dynamics simulations and quantum‐mechanics calculations for Complex II:FAD and Complex II:FADH2, with and without the covalent bond, were performed. It was observed that the covalent bond is essential for the active‐center arrangement of the FADH2/FAD cofactor. Removal of this bond causes a displacement of the isoalloxazine group, which influences interactions with the protein, flavin solvation, and possible proton‐transfer pathways. Specifically, for the noncovalently bound FADH2 cofactor, the N1 atom moves away from the His‐A365 and His‐A254 residues and the N5 atom moves away from the glutamine‐62A residue. Both of the histidine and glutamine residues interact with a chain of water molecules that cross the enzyme, which is most likely involved in proton transfer. Breaking this chain of water molecules could thereby compromise proton transfer across the two active sites of Complex II.