COMPUTATIONAL MODELING OF S-THIOLATION REACTION: TOWARD DISCOVERING THE ENZYMATIC MECHANISMS OF ENDOGENOUS HNO FORMATION Elena V. Ivanova, B.S. Marquette University, 2017 S-nitrosothiols (RSNOs) have long been proposed as potential sources of the elusive endogenous nitroxyl (HNO) via S-thiolation reaction with thiols. It is however not clear how S-thiolation can compete with the trans-S-nitrosation pathway commonly observed in vitro. Based on the insights into the highly unusual, antagonistic chemical nature of RSNO molecules, we hypothesize that, while difficult in vitro, S-thiolation could easily lend itself to enzymatic catalysis. To explore this possibility, we adopted a bottom-up computational approach that aims to identify possible catalytic mechanisms able to steer the RSNO + thiol reaction toward HNO production. Inspired by recent discovery of small bioactive HSNO molecule and its involvement in HNO production, we first study HSNO molecule and its subsequent isomerization reactions that can possibly lead to other potentially bioactive small molecules or reactive intermediates that can undergo S-thiolation reaction. Then, we mapped out the profile of the uncatalysed Sthiolation reaction that suggests that the most difficult step of S-thiolation is the R’SH to RSNO proton transfer, and that the reaction proceeds through unusual highly polar zwitterionic species R’SS(R)N(H)O that further decomposes yielding HNO and disulfide RSSR’. Therefore, facilitating the proton transfer and stabilizing this zwitterionic intermediate could lead to dramatic acceleration of this reaction. Further, we identified the required arrangements of amino acid residues using theozyme (‘theoretical enzyme’) modeling. These calculations showed that several of these putative active site models can drop the energetic barrier for S-thiolation/HNO formation to less than 10 kcal/mol. An extensive search in the RCSB protein data bank yielded over 600 structures that match one of these successful theozyme models. Remarkably, among these proteins we found DJ-1 protein known to be involved in RSNO-related processes. Furthermore, full-scale modeling of S-thiolation between an incoming RSNO and DJ-1 Cys106 using a hybrid quantum mechanics/molecular mechanics approach have shown that the reaction can be indeed efficiently catalyzed by the His126–Glu18 dyad, a prediction whose relevance to the DJ-1/RSNO biochemistry presents an intriguing question to the experimentalists.

Computational Modeling of S-Thiolation Reaction: Toward Discovering the Enzymatic Mechanisms of Endogenous HNO Formation

Ce travail s’inscrit dans l’etude de l’incidence de siderophores sur la physiologie de la plante. Il decrit plus precisement, a l’echelle phenotypique et moleculaire, l’impact de la pyoverdine produite par la souche bacterienne benefique Pseudomonas fluorescens C7R12 sur la croissance, la reponse immunitaire et l’homeostasie du fer chez Arabidopsis thaliana. Le lien fonctionnel entre immunite et homeostasie du fer a ete aborde de facon plus specifique via l’analyse du mode d’action de l’acide β-aminobutyrique (BABA), un potentialisateur des reponses de defense de la plante.En conditions de fer limitantes, afin de pourvoir a la carence, Pseudomonas fluorescens libere la pyoverdine dans le sol sous sa forme non chelatee (apo-pyo). Le complexe fer-pyoverdine (ferri-pyo) est ensuite internalise par la bacterie. Nous avons verifie que l’apo-pyo est assimilee par des plantes d’A. thaliana cultivees dans un milieu contenant ou non du fer. De facon remarquable, l’apo-pyo restaure le phenotype de croissance des plantes carencees en fer. Une analyse transcriptomique a revele que chez ces dernieres, l’apo-pyo induit fortement l’expression de genes associes a la croissance, l’import et la redistribution du fer in planta. En revanche, une repression de l’expression de genes de defense s’opere. De facon concordante, l’effet promoteur de croissance de l’apo-pyo chez les plantes carencees est strictement dependant de l’expression des genes IRT1 et FRO2 codant deux proteines majeures de l’import de fer. De plus, une moindre resistance de ces plantes a Botrytis cinerea a ete relevee. L’incidence negative de l’apo-pyo sur les defenses s’accompagne d’une surexpression du facteur de transcription HBI1 jouant un role cle dans la regulation de la balance croissance/defense. L’ensemble de ces evenements n’a pas ete observe chez les plantes cultivees dans un milieu enrichi en fer, demontrant que les effets de l’apo-pyo chez A. thaliana sont conditionnes par le statut en fer de la plante. En parallele, l’etude du mode d’action du BABA a indique que ce potentialisateur de l’immunite est un chelateur tres efficace du fer. En consequence, applique a des plantes d’A. thaliana, le BABA declenche une carence en fer transitoire. Nous avons emis l’hypothese que cette carence pourrait constituer un signal placant la plante en veille defensive. En accord avec cette assomption, les plantes carencees en fer presentent une resistance accrue a B. cinerea et produisent des metabolites secondaires associes aux defenses dont l’accumulation est egalement induite par le BABA. Ainsi, la carence en fer transitoire occasionnee par le BABA pourrait constituer l’une des composantes de son effet potentialisateur sur l’immunite. En conclusion, ce travail apporte des premiers elements explicatifs quant a l’incidence de la pyoverdine sur des traits physiologiques de la plante et rapporte un mode d’action orignal du BABA. Plus generalement, il renforce le concept encore naissant de l’existence de regulations croisees entre les voies de signalisation associees a la croissance, l’immunite et l’homeostasie du fer chez les plantes.

https://tel.archives-ouvertes.fr/tel-01418768/document

Incidence physiologique et étude du mode d'action de la pyoverdine de Pseudomonas fluorescens chez Arabidopsis thaliana : liens avec l'homéostasie du fer, la croissance et les défenses

ON THE STRUCTURE AND PROPERTIES OF S-NITROSATED CYSTEINE MODELS: A COMPUTATIONAL STUDY Dmitry G. Khomyakov, B.Sc., M.Sc. Marquette University, 2017 S-Nitrosation of cysteine (Cys) residues, a covalent modification of its S atom by NO group, is a major post-translational modification of proteins. Despite the importance of S-nitrosoproteins in numerous physiological processes, lability of the S-nitrosothiol (-SNO) group hinders the research progress. In this work, computational chemistry methods were applied to S-nitrosated cysteine (CysNO) models to gain a deeper insight into its structure and properties. First, we obtained the most accurate at the moment computational estimation of the molecular structure and properties of CH3SNO model molecule using Feller-Peterson-Dixon (FPD) ab initio protocol. The S–N bond length in cisCH3SNO is calculated as 1.814 Å, and its dissociation energy (BDE) is 32.4 kcal/mol. We found that although the vibrational frequency of the S–N stretch is unusually low for a covalent bond (398 cm-1), the S–N bond has a remarkably harmonic character. After the benchmarking of the density functional theory (DFT) methods against the FPD reference, we recommend MPW2PLYP and MPW2PLYPD double hybrid functionals for calculation of the geometric properties, vibrational frequencies and isomerization barriers of S-nitrosothiols, and PBE0 (PBE0-GD3) hybrid functional for the S–N BDEs. Further, we evaluated the influence of charged amino acid residues and steric constraints on the conformational dynamics of CysNO, using an a-helix model and a DJ-1 (PARK7) protein using hybrid quantum mechanics/molecular mechanics (QM/MM) approach. We found that while the rotational barrier around the S–N bond is ca. 13 kcal/mol in free CysNO, it can vary between 10 and 24 kcal/mol in the protein environment due to the effect of neighboring charged amino acids. Finally, to address a long-standing problem of the CysNO identification in proteins, we computationally designed a novel CysNO labeling reaction, (3+2) dipolar cycloaddition between the -SNO group activated by N-coordinated Lewis acid and a strain-activated alkene. We show that the most effective labeling reagent should covalently link both crucial reaction components—the Lewis acid and the dipolarophile—into one molecule, to lower the entropic penalty and corresponding reaction barrier.

On the Structure and Properties of S-Nitrosated Cysteine Models: A Computational Study

Nitric oxide (NO) is a free radical with a signaling capacity. Its cellular functions are achieved mainly through S-nitrosation where thioredoxin (hTrx) is pivotal in the S-transnitrosation to specific cellular targets. In this study, we use NMR spectroscopy and mass spectrometry to follow the mechanism of S-(trans)nitrosation of hTrx. We describe a site-specific path for S-nitrosation by measuring the reactivity of each of the 5 cysteines of hTrx using cysteine mutants. We showed the interdependence of the three cysteines in the nitrosative site. C73 is the most reactive and is responsible for all S-transnitrosation to other cellular targets. We observed NO internal transfers leading to C62 S-nitrosation, which serves as a storage site for NO. C69-SNO only forms under nitrosative stress, leading to hTrx nuclear translocation.

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Deciphering the Path of S-nitrosation of Human Thioredoxin: Evidence of an Internal NO Transfer and Implication for the Cellular Responses to NO

Reactive Sulfur Species (RSS), such as allicin from garlic or sulforaphane from broccoli, are fre-quently associated with biological activities and possible health benefits in animals and humans. Among these Organic Sulfur Compounds (OSCs) found in many plants and fungi, the Volatile Sulfur Compounds (VSCs) feature prominently, not only because of their often-pungent smell, but also because they are able to access places which solids and solutions cannot reach that easily. Indeed, inorganic RSS such as hydrogen sulfide (H2S) and sulfur dioxide (SO2) can be used to lit-erally fumigate entire rooms and areas. Similarly, metabolites of garlic, such as allyl methyl sulfide (AMS), are formed metabolically in humans in lower concentrations and reach the airways from inside the body as part of one’s breath. Curiously, H2S is also formed in the gastrointestinal tract by gut bacteria, and the question of if and for which purpose this gas then crosses the barriers and enters the body is indeed a delicate matter for equally delicate studies. In any case, nature is surprisingly rich in such VSCs, as fruits (for instance, the infamous durian) demonstrate, and therefore these VSCs represent a promising group of compounds for further studies.

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A Whiff of Sulfur: One Wind a Day Keeps the Doctor Away

There is currently great interest in S-nitrosothiols (RSNOs) because formation of protein-based RSNOsprotein S-nitrosationhas been recently recognized as a major pathway of the biological function of nitric oxide, NO. Despite the growing number of S-nitrosated proteins identified in vivo, enzymatic processes that control reactions of biological RSNOs are still not well understood. In this article, we use a range of models to computationally demonstrate that specific interactions of RSNOs with charged and polar residues in proteins can result in dramatic modification of RSNO structure, stability, and reactivity. This unprecedented sensitivity of the −SNO group toward interactions with charged species is related to their unusual electronic structure that can be elegantly expressed in terms of antagonistic resonance structures. We propose a ‘ligand effect map’ (LEM) approach as an efficient way to estimate the environment effects on the −SNO groups in proteins without performing electronic structure calculations. Furthermore, the calculated ¹⁵N NMR signatures of these specific interactions suggest that ¹⁵N NMR spectroscopy can be an effective technique to identify and study these interactions experimentally. Overall, the results of this study suggest that RSNO reactions in vivo should be tightly controlled by the protein environment via modulation of the RSNO electronic structure.

MaratR. Talipov

Papers

Protein Control of S-Nitrosothiol Reactivity: Interplay of Antagonistic Resonance Structures B