Catalytic iron-carbene intermediate revealed in a cytochromeccarbene transferase

Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostableRhodothermus marinus(Rma) cytochromec. High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and how the laboratory-evolved enzyme achieves perfect carbene transfer stereoselectivity by holding the catalytic IPC in a single orientation. We also discovered that the IPC inRmacytochromechas a singlet ground electronic state and that the protein environment uses geometrical constraints and noncovalent interactions to influence different IPC electronic states. This information helps us to understand the impressive reactivity and selectivity of carbene transfer enzymes and offers insights that will guide and inspire future engineering efforts.

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Design of an in vitro biocatalytic cascade for the manufacture of islatravir

Science ◽

10.1126/science.aay8484 ◽

2019 ◽

Vol 366 (6470) ◽

pp. 1255-1259 ◽

Cited By ~ 67

Author(s):

Mark A. Huffman ◽

Anna Fryszkowska ◽

Oscar Alvizo ◽

Margie Borra-Garske ◽

Kevin R. Campos ◽

...

Keyword(s):

Chemical Synthesis ◽

Directed Evolution ◽

Building Blocks ◽

Hiv Treatment ◽

Pharmaceutical Manufacturing ◽

Enzymatic Reactions ◽

Catalyzed Reactions ◽

Enzyme Catalyzed Reactions

Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.

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A kinetic analysis of coupled sequential enzyme reactions

10.26434/chemrxiv.5746065.v2 ◽

2018 ◽

Author(s):

Justin Eilertsen ◽

Santiago Schnell

Keyword(s):

Enzyme Assay ◽

Sequential Reaction ◽

Enzyme Reactions ◽

Indicator Reaction ◽

Single Substrate ◽

Complex Sequences ◽

Catalyzed Reactions ◽

Enzyme Catalyzed Reactions ◽

Single Enzyme

<div>As a case study, we consider a coupled enzyme assay of sequential enzyme reactions obeying the Michaelis--Menten reaction mechanism. The sequential reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction becomes the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the sequential reaction is described by a pair of interacting Michaelis--Menten equations. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis--Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

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A kinetic analysis of coupled sequential enzyme reactions

10.26434/chemrxiv.5746065.v1 ◽

2018 ◽

Author(s):

Justin Eilertsen ◽

Santiago Schnell

Keyword(s):

Enzyme Assay ◽

Sequential Reaction ◽

Enzyme Reactions ◽

Indicator Reaction ◽

Single Substrate ◽

Complex Sequences ◽

Catalyzed Reactions ◽

Enzyme Catalyzed Reactions ◽

Single Enzyme

<div>As a case study, we consider a coupled enzyme assay of sequential enzyme reactions obeying the Michaelis-Menten reaction mechanism. The sequential reaction consists of a single-substrate, single enzyme non-observable reaction followed by another single-substrate, single enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction becomes the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the sequential reaction is described by a pair of interacting Michaelis-Menten equations. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis-Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

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Computationally Augmented Retrosynthesis: Total Synthesis of Paspaline A and Emindole PB

10.26434/chemrxiv.7322330.v1 ◽

2018 ◽

Author(s):

Timothy Newhouse ◽

Daria E. Kim ◽

Joshua E. Zweig

Keyword(s):

Chemical Synthesis ◽

Total Synthesis ◽

Small Molecules ◽

First Principles ◽

Quantum Chemical ◽

Computational Analysis ◽

Empirical Evaluation ◽

Synthetic Strategy ◽

Catalyzed Reactions ◽

Enzyme Catalyzed Reactions

The diverse molecular architectures of terpene natural products are assembled by exquisite enzyme-catalyzed reactions. Successful recapitulation of these transformations using chemical synthesis is hard to predict from first principles and therefore challenging to execute. A means of evaluating the feasibility of such chemical reactions would greatly enable the development of concise syntheses of complex small molecules. Herein, we report the computational analysis of the energetic favorability of a key bio-inspired transformation, which we use to inform our synthetic strategy. This approach was applied to synthesize two constituents of the historically challenging indole diterpenoid class, resulting in a concise route to (–)-paspaline A in 9 steps from commercially available materials and the first pathway to and structural confirmation of emindole PB in 13 steps. This work highlights how traditional retrosynthetic design can be augmented with quantum chemical calculations to reveal energetically feasible synthetic disconnections, minimizing time-consuming and expensive empirical evaluation.

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Strongly Coupled Redox-Linked Conformational Switching at the Active Site of the Non-Heme Iron-Dependent Dioxygenase, TauD

10.26434/chemrxiv.9461462 ◽

2019 ◽

Author(s):

Christopher John ◽

Greg M. Swain ◽

Robert P. Hausinger ◽

Denis A. Proshlyakov

Keyword(s):

Active Site ◽

Structural Model ◽

Heme Iron ◽

Chemical Transformations ◽

Experimental Conditions ◽

Strongly Coupled ◽

Non Heme Iron ◽

Reversible Redox ◽

Wide Range ◽

Complex Structural

2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. We characterize an <i>in situ</i> structural model of the putative transient ferric intermediate of 2OG:taurine dioxygenase (TauD) by using a combination of spectroelectrochemical and semi-empirical computational methods, demonstrating that the Fe (III/II) transition involves a substantial, fully reversible, redox-linked conformational change at the active site. This rearrangement alters the apparent redox potential of the active site between -127 mV for reduction of the ferric state and 171 mV for oxidation of the ferrous state of the 2OG-Fe-TauD complex. Structural perturbations exhibit limited sensitivity to mediator concentrations and potential pulse duration. Similar changes were observed in the Fe-TauD and taurine-2OG-Fe-TauD complexes, thus attributing the reorganization to the protein moiety rather than the cosubstrates. Redox difference infrared spectra indicate a reorganization of the protein backbone in addition to the involvement of carboxylate and histidine ligands. Quantitative modeling of the transient redox response using two alternative reaction schemes across a variety of experimental conditions strongly supports the proposal for intrinsic protein reorganization as the origin of the experimental observations.

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Organocatalyzed Heterocyclic Transformations In Green Media: A Review

Current Organocatalysis ◽

10.2174/2213337207999200805115813 ◽

2020 ◽

Vol 07 ◽

Author(s):

Neslihan Demirbas ◽

Ahmet Demirbas

Keyword(s):

Hydrogen Bonding ◽

Phase Transfer ◽

Noncovalent Interactions ◽

Binding Capacity ◽

Chemical Transformations ◽

Organic Transformations ◽

Phase Transfer Catalysts ◽

Waste Production ◽

Synthetic Methodologies ◽

Organocatalytic Reactions

Background: Since the discovery of metal-free catalysts or organocatalysts about twenty years ago, a number of small molecules with different structures have been using to accelerate organic transformations. With the development of environmental awareness, in order to obtain highly privileged scaffolds, scientists have directed their studies towards the synthetic methodologies which minimize or preferably eliminate the formation of waste, avoid from toxic solvents and reagents and use renewable starting materials as far as possible. Methods: In this connection, the organocatalytic reactions providing efficiency and selectivity for most of case have become an endless topic in organic chemistry since several advantages from both practical and environmental standpoints. Organocatalysts supplying transformation of reactants into products with the least possible waste production have been serving to the concept of green chemistry. Results and Conclusion: Organocatalysts have been classified on the basis of their binding capacity to the substrate with covalently or noncovalent interactions involving hydrogen bonding and electrostatic interaction. Diverse types of small organic compounds including proline and its derivatives, phase-transfer catalysts, (thio)urease, phosphoric acids, sulfones, N-oxides, guanidines, cinchona derivatives, aminoindanol and amino acids have been utilized as hydrogen bonding organocatalysts in different chemical transformations.

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Diverse Taxonomies for Diverse Chemistries: Enhanced Representation of Natural Product Metabolism in UniProtKB

Metabolites ◽

10.3390/metabo11010048 ◽

2021 ◽

Vol 11 (1) ◽

pp. 48

Author(s):

Marc Feuermann ◽

Emmanuel Boutet ◽

Anne Morgat ◽

Kristian Axelsen ◽

Parit Bansal ◽

...

Keyword(s):

Natural Products ◽

Natural Product ◽

Semantic Web Technologies ◽

Knowledge Resources ◽

Web Technologies ◽

Chemical Structures ◽

Biological Interest ◽

Catalyzed Reactions ◽

Enzyme Catalyzed Reactions ◽

Practical Demonstration

The UniProt Knowledgebase UniProtKB is a comprehensive, high-quality, and freely accessible resource of protein sequences and functional annotation that covers genomes and proteomes from tens of thousands of taxa, including a broad range of plants and microorganisms producing natural products of medical, nutritional, and agronomical interest. Here we describe work that enhances the utility of UniProtKB as a support for both the study of natural products and for their discovery. The foundation of this work is an improved representation of natural product metabolism in UniProtKB using Rhea, an expert-curated knowledgebase of biochemical reactions, that is built on the ChEBI (Chemical Entities of Biological Interest) ontology of small molecules. Knowledge of natural products and precursors is captured in ChEBI, enzyme-catalyzed reactions in Rhea, and enzymes in UniProtKB/Swiss-Prot, thereby linking chemical structure data directly to protein knowledge. We provide a practical demonstration of how users can search UniProtKB for protein knowledge relevant to natural products through interactive or programmatic queries using metabolite names and synonyms, chemical identifiers, chemical classes, and chemical structures and show how to federate UniProtKB with other data and knowledge resources and tools using semantic web technologies such as RDF and SPARQL. All UniProtKB data are freely available for download in a broad range of formats for users to further mine or exploit as an annotation source, to enrich other natural product datasets and databases.

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