scholarly journals An In Vivo Biocatalytic Cascade Featuring an Artificial Enzyme Catalyzed New-to-Nature Reaction

2021 ◽  
Author(s):  
Linda Ofori Atta ◽  
Zhi Zhou ◽  
Gerard Roelfes
Keyword(s):  
Carbonyl Compounds ◽  
Artificial Enzymes ◽  
Catalytic Residue ◽  
Artificial Enzyme ◽  
E Coli ◽  
Iminium Ion ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Benzaldehyde Derivatives

Artificial enzymes utilizing the genetically encoded non-proteinogenic amino acid p-aminophenylalanine (pAF) as catalytic residue are able to react with carbonyl compounds through an iminium ion mechanism, making reactions possible that have no equivalent in nature. Here, we report an in vivo biocatalytic cascade that is augmented with such an artificial enzyme catalyzed new-to-nature reaction. The artificial enzyme in this study is a pAF containing evolved variant of the Lactococcal multidrug resistance Regulator, designated LmrR_V15pAF_RMH, which efficiently converts in vivo produced benzaldehyde derivatives into the corresponding hydrazone products inside E. coli cells. These in vivo biocatalytic cascades comprising an artificial enzyme catalyzed reactions are an important step towards achieving a hybrid metabolism.

scholarly journals Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase

2019 ◽  
Vol 60 (5) ◽  
pp. 953-962 ◽  
Author(s):  
Peter J. Harrison ◽  
Kenneth Gable ◽  
Niranjanakumari Somashekarappa ◽  
Van Kelly ◽  
David J. Clarke ◽  
...  
Keyword(s):  
In Vivo Studies ◽  
Serine Palmitoyltransferase ◽  
Biochemical Pathways ◽  
Catalytic Mechanisms ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
The Impact ◽  
Sphingolipid Biosynthesis

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis. Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.

Some enzymic syntheses of 15N-L-aspartic acid and 15N-L-glutamic acid

1969 ◽  
Vol 47 (3) ◽  
pp. 411-415 ◽  
Author(s):  
J. A. Zintel ◽  
A. J. Williams ◽  
R. S. Stuart
Keyword(s):  
Glutamic Acid ◽  
Aspartic Acid ◽  
Fumaric Acid ◽  
Aspartate Aminotransferase ◽  
Efficient Utilization ◽  
E Coli ◽  
Acid Dehydrogenase ◽  
Acid Catalyzed ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

Some methods for the preparation of 15N-L-aspartic acid and 15N-L-glutamic acid using enzyme catalyzed reactions are described. 15N-L-Aspartic acid is prepared by the addition of ammonia to fumaric acid, catalyzing the reaction with aspartase which was partially purified from E. coli. 15N-L-Glutamic acid with a high 15N/14N ratio is prepared by transamination with 15N-L-aspartic acid catalyzed by aspartate aminotransferase. 15N-L-Glutamic acid with a low 15N/14N ratio is prepared by a glutamic acid dehydrogenase catalyzed exchange of L-glutamic acid with 15N-ammonia. These enzymes are commercially available. Since efficient utilization of 15N is obtained, aspartic acid or glutamic acid may be prepared with any desired 15N/14N ratio.

scholarly journals Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel-Crafts Alkylase

2021 ◽  
Author(s):  
Reuben B. Leveson-Gower ◽  
Zhi Zhou ◽  
Ivana Drienovská ◽  
Gerard Roelfes
Keyword(s):  
Artificial Enzymes ◽  
Artificial Enzyme ◽  
Triple Mutant ◽  
Protein Scaffold ◽  
Rate Acceleration ◽  
Iminium Ion ◽  
Canonical Amino Acid ◽  
Catalytic Reactivity ◽  
Ion Species ◽  
Kinetics Of

We show that the incorporation of the non-canonical amino acid para-aminophenylalanine (pAF) into the non-enzymatic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel-crafts alkylation between alpha, beta-unsaturated aldehydes and indoles. pAF acts as a catalytic residue, activating enal substrates towards conjugate addition via the formation of intermediate iminium ion species, whilst the protein scaffold provides rate acceleration and enantio-induction. Improved LmrR_pAF varants were identified by direted evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of enals and indoles. Analys of Michaelis-Menten kinetics of LmrR-pAF and tevolved mutants reveals that new activities emerge via evolutionary pathways that diverge from one another and specialise catalytic reactivity.<br>

scholarly journals Development of a vanillate biosensor for the vanillin biosynthesis pathway in E. coli

2018 ◽  
Author(s):  
Aditya M. Kunjapur ◽  
Kristala L. J. Prather
Keyword(s):  
Metabolic Engineering ◽  
Dynamic Range ◽  
De Novo ◽  
Caulobacter Crescentus ◽  
Homo Sapiens ◽  
Biosynthesis Pathway ◽  
Dynamic Regulation ◽  
E Coli ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

AbstractGenetically encoded small molecule sensors can facilitate metabolic engineering by enabling high-throughput detection of metabolite concentrations, directed evolution of host and pathway enzymes, and dynamic regulation. The engineered de novo vanillin biosynthesis pathway assembled in Escherichia coli is industrially relevant and ideal for biosensor deployment given that the pathway requires only three heterologous enzyme-catalyzed reactions, generates naturally occurring metabolites, and may benefit from dynamic regulation. However, pathway flux is stalled and diverted by the activity of the Homo sapiens catechol O-methyltransferase, which is intended to catalyze the conversion of protocatechuate to vanillate. To confront this challenge, we constructed and applied a vanillate sensor based on the Caulobacter crescentus VanR-VanO system. Using components from a previously characterized E. coli promoter library, we achieved greater than 14-fold dynamic range in our best rationally constructed sensor. We characterized sensor substrate specificity and found that this construct and an evolved variant are remarkably selective, exhibiting no detectable response to the regioisomer byproduct isovanillate. We then harnessed the evolved biosensor to conduct rapid bioprospecting of natural catechol O-methyltransferases. We identified eight that appear to have greater desired activity than the originally used variant, including three previously uncharacterized O-methyltransferases. Collectively, these efforts enrich our knowledge of how biosensing can aid metabolic engineering and constitute the foundation for future improvements in vanillin pathway productivity.

scholarly journals Pic, an Autotransporter Protein Secreted by Different Pathogens in the Enterobacteriaceae Family, Is a Potent Mucus Secretagogue

2010 ◽  
Vol 78 (10) ◽  
pp. 4101-4109 ◽  
Author(s):  
Fernando Navarro-Garcia ◽  
Javier Gutierrez-Jimenez ◽  
Carlos Garcia-Tovar ◽  
Luis A. Castro ◽  
Hector Salazar-Gonzalez ◽  
...  
Keyword(s):  
Serine Protease ◽  
Shigella Flexneri ◽  
Goblet Cells ◽  
Site Directed Mutagenesis ◽  
Catalytic Residue ◽  
Mucus Layer ◽  
Mucus Hypersecretion ◽  
E Coli ◽  
Intestinal Mucus

ABSTRACT A hallmark of enteroaggregative Escherichia coli (EAEC) infection is a formation of biofilm, which comprises a mucus layer with immersed bacteria in the intestines of patients. While studying the mucinolytic activity of Pic in an in vivo system, rat ileal loops, we surprisingly found that EAEC induced hypersecretion of mucus, which was accompanied by an increase in the number of mucus-containing goblet cells. Interestingly, an isogenic pic mutant (EAEC Δpic) was unable to cause this mucus hypersecretion. Furthermore, purified Pic was also able to induce intestinal mucus hypersecretion, and this effect was abolished when Pic was heat denatured. Site-directed mutagenesis of the serine protease catalytic residue of Pic showed that, unlike the mucinolytic activity, secretagogue activity did not depend on this catalytic serine protease motif. Other pathogens harboring the pic gene, such as Shigella flexneri and uropathogenic E. coli (UPEC), also showed results similar to those for EAEC, and construction of isogenic pic mutants of S. flexneri and UPEC confirmed this secretagogue activity. Thus, Pic mucinase is responsible for one of the pathophysiologic features of the diarrhea mediated by EAEC and the mucoid diarrhea induced by S. flexneri.

scholarly journals Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel-Crafts Alkylase

2021 ◽  
Author(s):  
Reuben B. Leveson-Gower ◽  
Zhi Zhou ◽  
Ivana Drienovská ◽  
Gerard Roelfes
Keyword(s):  
Artificial Enzymes ◽  
Artificial Enzyme ◽  
Triple Mutant ◽  
Protein Scaffold ◽  
Rate Acceleration ◽  
Iminium Ion ◽  
Canonical Amino Acid ◽  
Catalytic Reactivity ◽  
Ion Species ◽  
Kinetics Of

We show that the incorporation of the non-canonical amino acid para-aminophenylalanine (pAF) into the non-enzymatic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel-crafts alkylation between alpha, beta-unsaturated aldehydes and indoles. pAF acts as a catalytic residue, activating enal substrates towards conjugate addition via the formation of intermediate iminium ion species, whilst the protein scaffold provides rate acceleration and enantio-induction. Improved LmrR_pAF varants were identified by direted evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of enals and indoles. Analys of Michaelis-Menten kinetics of LmrR-pAF and tevolved mutants reveals that new activities emerge via evolutionary pathways that diverge from one another and specialise catalytic reactivity.<br>

scholarly journals In vitro biosynthesis of ATP from adenosine and polyphosphate

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Chuanqi Sun ◽  
Zonglin Li ◽  
Xiao Ning ◽  
Wentian Xu ◽  
Zhimin Li
Keyword(s):  
Adenosine Kinase ◽  
Inorganic Polyphosphate ◽  
One Pot ◽  
Atp Production ◽  
Wide Ph Range ◽  
Catalyzed Reactions ◽  
In Vitro Biosynthesis ◽  
Enzyme Catalyzed Reactions

AbstractAdenosine triphosphate (ATP) acts as a crucial energy currency in vivo, and it is a widely used energy and/or phosphate donor for enzyme-catalyzed reactions in vitro. In this study, we established an in vitro multi-enzyme cascade system for ATP production. Using adenosine and inorganic polyphosphate (polyP) as key substrates, we combined adenosine kinase and two functionally distinct polyphosphate kinases (PPKs) in a one-pot reaction to achieve chain-like ATP regeneration and production. Several sources of PPK were screened and characterized, and two suitable PPKs were selected to achieve high rates of ATP production. Among these, Sulfurovum lithotrophicum PPK (SlPPK) exhibited excellent activity over a wide pH range (pH 4.0–9.0) and synthesized ATP from ADP using short-chain polyP. Furthermore, it had a half-life > 155.6 h at 45 °C. After optimizing the reaction conditions, we finally carried out the coupling-catalyzed reaction with different initial adenosine concentrations of 10, 20, and 30 mM. The highest yields of ATP were 76.0, 70.5, and 61.3%, respectively. Graphical Abstract

Fe-N-C Artificial Enzyme: Activation of Oxygen for Dehydrogenation and Monoxygenation of Organic Substrates under Mild Condition and Cancer Therapeutic Application

2018 ◽  
Author(s):  
Fei He ◽  
Li Mi ◽  
Yanfei Shen ◽  
Toshiyuki Mori ◽  
Songqin Liu ◽  
...  
Keyword(s):  
Mild Condition ◽  
Enzyme Activation ◽  
Artificial Enzymes ◽  
Organic Substrates ◽  
Artificial Enzyme ◽  
Therapeutic Application ◽  
Highly Efficient ◽  
Activation Of Oxygen

Developing highly efficient artificial enzymes that directly employ O<sub>2</sub> as terminal oxidant has long been pursued but has rarely achieved yet. We report Fe-N-C has unusual enzyme-like activity in both dehydrogenation and monoxygenation of organic substrates with ~100% selectivity by direct using O<sub>2</sub>.

A kinetic analysis of coupled sequential enzyme reactions

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>

A kinetic analysis of coupled sequential enzyme reactions

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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