scholarly journals Directed Evolution of a Ketone Synthase for Efficient and Highly Selective Functionalization of Internal Alkenes by Accessing Reactive Carbocation Intermediates

2022 ◽  
Author(s):  
Sebastian Gergel ◽  
Jordi Soler ◽  
Alina Klein ◽  
Kai Schülke ◽  
Bernhard Hauer ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Small Molecule ◽  
Active Site ◽  
Computational Analysis ◽  
Catalytic Cycle ◽  
Enantioselective Oxidation ◽  
Dynamical Network ◽  
Selective Functionalization ◽  
Synthetic Routes ◽  
Regioselective Oxidation

The direct regioselective oxidation of internal alkenes to ketones could simplify synthetic routes and solve a longstanding challenge in synthesis. This reaction is of particular importance because ketones are predominant moieties in valuable products as well as crucial intermediates in synthesis. Here we report the directed evolution of a ketone synthase that oxidizes internal alkenes directly to ketones with several thousand turnovers. The evolved ketone synthase benefits from more than a dozen crucial mutations, most of them distal to the active site. Computational analysis reveals that all these mutations collaborate to facilitate the formation of a highly reactive carbocation intermediate by generating a confined, rigid and preorganized active site through an enhanced dynamical network. The evolved ketone synthase fully exploits a catalytic cycle that has largely eluded small molecule catalysis and consequently enables various challenging functionalization reactions of internal alkenes. This includes the first catalytic, enantioselective oxidation of internal alkenes to ketones, as well as the formal asymmetric hydration and hydroamination of unactivated internal alkenes in combination with other biocatalysts.

scholarly journals Local frustration determines loop opening during the catalytic cycle of an oxidoreductase

2019 ◽  
Author(s):  
Lukas L. Stelzl ◽  
Despoina A.I. Mavridou ◽  
Emmanuel Saridakis ◽  
Diego Gonzalez ◽  
Andrew J. Baldwin ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Protein Function ◽  
Catalytic Cycle ◽  
Biomolecular Recognition ◽  
Competing Interactions ◽  
Protein Binding Sites ◽  
Loop Region ◽  
Cysteine Thiols ◽  
Small Chemical

AbstractLocal structural frustration, the existence of mutually exclusive competing interactions, may explain why some proteins are dynamic while others are rigid. Frustration is thought to underpin biomolecular recognition and the flexibility of protein binding sites. Here we show how a small chemical modification, the oxidation of two cysteine thiols to a disulfide bond, during the catalytic cycle of the N-terminal domain of the key bacterial oxidoreductase DsbD (nDsbD), introduces frustration ultimately influencing protein function. In oxidized nDsbD, local frustration disrupts the packing of the protective cap-loop region against the active site allowing loop opening. By contrast, in reduced nDsbD the cap loop is rigid, always protecting the active-site thiols from the oxidizing environment of the periplasm. Our results point towards an intricate coupling between the dynamics of the active-site cysteines and of the cap loop which modulates the association reactions of nDsbD with its partners resulting in optimized protein function.

scholarly journals Enhancing a De Novo Enzyme Activity by Computationally-Focused, Ultra-Low-Throughput Sequence Screening

2020 ◽  
Author(s):  
Valeria A. Risso ◽  
Adrian Romero-Rivera ◽  
Luis I. Gutierrez-Rus ◽  
Mariano Ortega-Muñoz ◽  
Francisco Santoyo-Gonzalez ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Active Site ◽  
De Novo ◽  
Enzymatic Reaction ◽  
Valence Bond ◽  
Computational Procedure ◽  
Enzyme Design ◽  
Computational Enzyme Design ◽  
Stability Ranking ◽  
Random Library

<div> <div> <div> <p>Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library screening remains a sluggish process, in large part due to futile probing of mutations that are catalytically neutral and/or impair stability and folding. FuncLib (funclib-weizmann.ac.il) is a novel automated computational procedure which uses phylogenetic analysis and Rosetta design to rank enzyme variants with multiple mutations, on the basis of a stability metric. Here, we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The similarity between the Michaelis complex and transition state for the enzymatic reaction makes this a particularly challenging system to optimize. Yet, experimental screening of a very small number of active-site, multi-point variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers (~2·104 M-1 s-1 and ~102 s-1) that compare well with modern natural enzymes, and that approach the catalysis levels for the best Kemp eliminases derived from extensive screening. This result illustrates the promise of FuncLib as a powerful tool with which to speed up directed evolution, by guiding screening to regions of the sequence space that encode stable and catalytically diverse enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the optimized eliminases to within ~2 kcal·mol-1 and indicate that the improvements in activity are linked to better geometric preorganization of the active site. This raises the possibility of further enhancing the stability-guidance of FuncLib by EVB-based computational predictions of catalytic activity, as a generalized approach for computational enzyme design. </p> </div> </div> </div>

Depsidone synthesis. XII. Some exploratory synthetic routes to highly functionalized depsidones

1978 ◽  
Vol 31 (6) ◽  
pp. 1383 ◽  
Author(s):  
T Sala ◽  
MV Sargent
Keyword(s):  
Selective Functionalization ◽  
Synthetic Routes

Attempts to synthesize the depsidones virensic acid (1), physciosporin (2), and pannarin (3) by routes based on the Ullmann ether condensation and the Hems reaction are described. Selective functionalization of highly substituted p-xylenes by photobromination is reported.

Abstract 523: Factor XIIa: New Insights on Chemical Tractability and Target Indications From a Potent and Selective Tool Inhibitor

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Christopher M Barbieri ◽  
Xinkang Wang ◽  
Xueping Zhou ◽  
Aimie M Ogawa ◽  
Kim O'Neil ◽  
...  
Keyword(s):  
Small Molecule ◽  
Active Site ◽  
Small Molecule Inhibitors ◽  
Plaque Rupture ◽  
Rabbit Model ◽  
Human Albumin ◽  
Stroke Treatment ◽  
Safe Strategy ◽  
Rupture Model

FXII is an emerging target for thrombosis, yet several questions remain to be addressed. Firstly, from drug discovery perspective, level of enzyme occupancy needed for efficacy (which largely dictates potency and selectivity requirement for small molecule inhibitors) is unclear, as most reported active site inhibitors have some level of off-target activities. Secondly, from disease treatment perspective, it is unclear whether FXIIa inhibition will be a safe strategy for stroke treatment or prevention, as it was recently reported that FXIIa inhibition destabilized the subocclusive thrombi in a plaque rupture model. In this presentation, we set out to address these questions using a previously described molecule, Inf4mut15. We generated the human albumin (HA)-Inf4mut15 fusion protein (Mut-inf) for our studies. In vitro Mut-inf displayed comparable potency as the widely used wild-type HA-Infestin4 (WT-inf) (human FXIIa Ki = 73 and 120 pM, respectively). Both infs acted as competitive reversible active site inhibitors of FXIIa, with no binding to FXII zymogen, hence same mode of action as certain small molecule inhibitors. Mut-inf, however, was much more selective against plasmin compared to WT-inf (20,000- and 75-fold Ki separation, respectively), consistent with results from the functional tPA-induced TEG assay, where Ly60 was reduced dose-dependently by WT- but not Mut-inf. Mut-inf aPTT doubling concentration was 15 uM and FXIIa Ki in 30% plasma was 3.5 nM. Calculated enzyme occupancy for Mut-inf for doubling human aPTT is thus 99.9%. In the rabbit model of cerebral microembolic signals (MES) induced by FeCl 3 injury of the carotid artery, treatment with vehicle (n=7), WT-, and Mut-inf (1mg/kg and n=5 each) produced arterial thrombus of 6.0±0.4, 1.9±0.6, and 0.2±0.1 mg, respectively; incidence of MES detected in the middle cerebral artery was 4.1±1.3, 1.8±0.6, and 0.0±0.0, respectively. In summary, our studies demonstrated that very high enzyme occupancy will be required for achieving a putative aPTT doubling target in human for FXIIa active site inhibitors, highlighting the challenge with the small molecule modality. Our MES studies suggest that targeting FXII may offer a safe strategy for stroke prevention and/or other thromboembolic disorders.

scholarly journals Confined acids catalyze asymmetric single aldolizations of acetaldehyde enolates

Science ◽  
2018 ◽  
Vol 362 (6411) ◽  
pp. 216-219 ◽  
Author(s):  
Lucas Schreyer ◽  
Philip S. J. Kaib ◽  
Vijay N. Wakchaure ◽  
Carla Obradors ◽  
Roberta Properzi ◽  
...  
Keyword(s):  
Small Molecule ◽  
Active Site ◽  
Aldol Reaction ◽  
Catalyst Loading ◽  
Mukaiyama Aldol Reaction ◽  
Mukaiyama Aldol

Reactions that form a product with the same reactive functionality as that of one of the starting compounds frequently end in oligomerization. As a salient example, selective aldol coupling of the smallest, though arguably most useful, enolizable aldehyde, acetaldehyde, with just one partner substrate has proven to be extremely challenging. Here, we report a highly enantioselective Mukaiyama aldol reaction with the simple triethylsilyl (TES) andtert-butyldimethylsilyl (TBS) enolates of acetaldehyde and various aliphatic and aromatic acceptor aldehydes. The reaction is catalyzed by recently developed, strongly acidic imidodiphosphorimidates (IDPi), which, like enzymes, display a confined active site but, like small-molecule catalysts, have a broad substrate scope. The process is scalable, fast, efficient (0.5 to 1.5 mole % catalyst loading), and greatly simplifies access to highly valuable silylated acetaldehyde aldols.

scholarly journals Computational analysis and predictive modeling of small molecule modulators of microRNA

2012 ◽  
Vol 4 (1) ◽  
Author(s):  
Salma Jamal ◽  
Vinita Periwal ◽  
OpenSourceDrugDiscovery Consortium ◽  
Vinod Scaria
Keyword(s):  
Small Molecule ◽  
Predictive Modeling ◽  
Computational Analysis ◽  
Small Molecule Modulators
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