DMSO-tolerant ornithine decarboxylase (ODC) tandem assay optimised for high-throughput screening

Park, Mingu Gordon; Kim, Suyeon Yellena; Lee, C. Justin

doi:10.1080/14756366.2022.2150186

Cited by 3 publications

(1 citation statement)

References 46 publications

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“…Ornithine decarboxylation is the rate-limiting step in polyamine biosynthesis [15]. Treatment with the irreversible ODC inhibitor difluoromethylornithine (DFMO) is known to deplete cellular levels of putrescine, spermidine, and agmatine [16,17].…”

Section: Introductionmentioning

confidence: 99%

Characterization of an Arginine Decarboxylase from Streptococcus pneumoniae by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

Lee,

Ayoola,

Shack

et al. 2024

Biomolecules

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Polyamines are polycations derived from amino acids that play an important role in proliferation and growth in almost all living cells. In Streptococcus pneumoniae (the pneumococcus), modulation of polyamine metabolism not only plays an important regulatory role in central metabolism, but also impacts virulence factors such as the capsule and stress responses that affect survival in the host. However, functional annotation of enzymes from the polyamine biosynthesis pathways in the pneumococcus is based predominantly on computational prediction. In this study, we cloned SP_0166, predicted to be a pyridoxal-dependent decarboxylase, from the Orn/Lys/Arg family pathway in S. pneumoniae TIGR4 and expressed and purified the recombinant protein. We performed biochemical characterization of the recombinant SP_0166 and confirmed the substrate specificity. For polyamine analysis, we developed a simultaneous quantitative method using hydrophilic interaction liquid chromatography (HILIC)-based liquid chromatography–tandem mass spectrometry (LC–MS/MS) without derivatization. SP_0166 has apparent Km, kcat, and kcat/Km values of 11.3 mM, 715,053 min−1, and 63,218 min−1 mM−1, respectively, with arginine as a substrate at pH 7.5. We carried out inhibition studies of SP_0166 enzymatic activity with arginine as a substrate using chemical inhibitors DFMO and DFMA. DFMO is an irreversible inhibitor of ornithine decarboxylase activity, while DFMA inhibits arginine decarboxylase activity. Our findings confirm that SP_0166 is inhibited by DFMA and DFMO, impacting agmatine production. The use of arginine as a substrate revealed that the synthesis of putrescine by agmatinase and N-carbamoylputrescine by agmatine deiminase were both affected and inhibited by DFMA. This study provides experimental validation that SP_0166 is an arginine decarboxylase in pneumococci.

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Section: Introductionmentioning

confidence: 99%

Characterization of an Arginine Decarboxylase from Streptococcus pneumoniae by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

Lee,

Ayoola,

Shack

et al. 2024

Biomolecules

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Dual‐Color Real‐Time Chemosensing of a Compartmentalized Reaction Network Involving Enzyme‐Induced Membrane Permeation of Peptides

Jiang,

Nilam,

Hennig

et al. 2023

Advanced Materials

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The design of synthetic systems with interrelated reaction sequences that model incipient biological complexity is limited by physicochemical tools that allow the direct monitoring of the individual processes in real time. To mimic a simple digestion‐resorption sequence, we have designed compartmentalized liposomal systems that incorporate extra‐ and intravesicular chemosensing ensembles. The extravesicular reporter pair consisted of cucurbit[7]uril and methylene blue to monitor the enzymatic cleavage of short enkephalin‐related peptides by thermolysin through a switch‐off fluorescence response (“digestion”). Because the substrate is membrane‐impermeable, but the dipeptide product permeable, an uptake of the latter into the pre‐formed liposomes occurred as follow‐up process. The intravesicular chemosensing ensemble consisted of (i) cucurbit[8]uril, 2‐anilinonaphthalene‐6‐sulfonic acid, and methyl viologen or (ii) cucurbit[7]uril and berberine to monitor the uptake (“resorption”) of the enzymatic products through the liposomal membranes by (i) a switch‐on or (ii) a switch‐off fluorescence response. The dyes were designed to allow selective optical excitation and read‐out of the extra‐ and intravesicular dyes, which allowed for a dual‐color chemosensing and, therefore, a kinetic discrimination of the two sequential reactions.This article is protected by copyright. All rights reserved

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A novel cascade catalysis for one-pot enzymatically modified isoquercitrin (EMIQ) conversion from rutin and sucrose using rationally designed gradient temperature control

Zhan,

Meng,

Yan

et al. 2024

Food Chemistry

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DMSO-tolerant ornithine decarboxylase (ODC) tandem assay optimised for high-throughput screening

Cited by 3 publications

References 46 publications

Characterization of an Arginine Decarboxylase from Streptococcus pneumoniae by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

Characterization of an Arginine Decarboxylase from Streptococcus pneumoniae by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

Dual‐Color Real‐Time Chemosensing of a Compartmentalized Reaction Network Involving Enzyme‐Induced Membrane Permeation of Peptides

A novel cascade catalysis for one-pot enzymatically modified isoquercitrin (EMIQ) conversion from rutin and sucrose using rationally designed gradient temperature control

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