Dataset Information

Selective Activation of N,N'-Diacyl Rhodamine Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).

ABSTRACT: This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine?110. We prepared a library of simple N,N'-diacyl rhodamines and investigated porcine liver esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluorescent upon PLE-mediated release. Rhodamine pro-fluorophore amides trifluoroacetamide (TFAm) and proponamide (PAm) appeared to be the best substrates using a cell-based assay using PLE expressing HEK293. Our pro-fluorophore series showed diffusion into live cells and resisted endogenous hydrolysis. The use of our engineered cell line containing the exogenous enzyme PLE demonstrated the rigorousness of amide masking when compared to cells not containing PLE. This simple and selective pro-fluorophore rhodamine pair with PLE offers the potential to be used in vitro and in vivo fluorescence based assays.

SUBMITTER: Abney KK

PROVIDER: S-EPMC6035858 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Selective Activation of N,N'-Diacyl Rhodamine Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).

Abney Kristopher K KK Ramos-Hunter Susan J SJ Romaine Ian M IM Goodwin J Shawn JS Sulikowski Gary A GA Weaver C David CD

Chemistry (Weinheim an der Bergstrasse, Germany) 20180525 36

This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine 110. We prepared a library of simple N,N'-diacyl rhodamines and investigated porcine liver esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluoresce ...[more]

PMID: 29679472

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Project description:BackgroundMicrobial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities.Methodology/principal findingsBacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2-C12) and triglycerides (C2-C6). Its optimal performance occurred at pH 8.5 and 50°C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6?/7? hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220.ConclusionEstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these enzymes showed hugely different thermal stabilities, indicating their diverse thermal adaptations via just changing a few amino acid residues.

Dataset Information

Selective Activation of N,N'-Diacyl Rhodamine Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).

Publications

Selective Activation of N,N'-Diacyl Rhodamine Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).

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